Nucleic acids encoding anti-C5a antibodies

ABSTRACT

The present disclosure relates to, inter alia, antibodies, or antigen-binding fragments thereof, that bind to C5a and to use of the antibodies in methods for treating or preventing complement-associated disorders such as, but not limited to, atypical hemolytic uremic syndrome, age-related macular degeneration, rheumatoid arthritis, sepsis, severe burn, antiphospholipid syndrome, asthma, lupus nephritis, Goodpasture&#39;s syndrome, and chronic obstructive pulmonary disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No.14/657,176, filed Mar. 13, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/695,277, filed Apr. 4, 2013, which is a nationalstage filing under 35 U.S.C. §371 of International Application No.PCT/US2011/034672, filed Apr. 29, 2011, which claims the benefit of thefiling date under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. Nos. 61/330,260, filed on Apr. 30, 2010, and61/471,465, filed on Apr. 4, 2011, the entire contents of which arehereby incorporated by reference in their entireties. InternationalApplication No. PCT/US2011/034672 was published under PCT Article 21(2)in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 4, 2015, is namedAXJ_154USDV2_Seq_List.txt, and is 143,968 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade progresses via the classical pathway, thealternative pathway, or the lectin pathway. These pathways share manycomponents, and while they differ in their initial steps, they convergeand share the same “terminal complement” components (C5 through C9)responsible for the activation and destruction of target cells.

The classical pathway (CP) is typically initiated by antibodyrecognition of, and binding to, an antigenic site on a target cell. Thealternative pathway (AP) can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. Additionally, thelectin pathway is typically initiated with binding of mannose-bindinglectin (MBL) to high mannose substrates. These pathways converge at thepoint where complement component C3 is cleaved by an active protease toyield C3a and C3b. Other pathways activating complement attack can actlater in the sequence of events leading to various aspects of complementfunction.

C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as wellas to certain viruses and immune complexes, and tags them for removalfrom the circulation. (C3b in this role is known as opsonin.) Theopsonic function of C3b is generally considered to be the most importantanti-infective action of the complement system. Patients with geneticlesions that block C3b function are prone to infection by a broadvariety of pathogenic organisms, while patients with lesions later inthe complement cascade sequence, i.e., patients with lesions that blockC5 functions, are found to be more prone only to Neisseria infection,and then only somewhat more prone.

C3b also forms a complex with other components unique to each pathway toform classical or alternative C5 convertase, which cleaves C5 into C5aand C5b. C3 is thus regarded as the central protein in the complementreaction sequence since it is essential to both the alternative andclassical pathways. This property of C3b is regulated by the serumprotease Factor I, which acts on C3b to produce iC3b. While stillfunctional as opsonin, iC3b cannot form an active C5 convertase.

C5 is a 190 kDa beta globulin found in normal serum at a concentrationof approximately 75 μg/mL (0.4 μM). C5 is glycosylated, with about 1.5to 3 percent of its mass attributed to carbohydrate. Mature C5 is aheterodimer of a 999 amino acid 115 kDa alpha chain that is disulfidelinked to a 655 amino acid 75 kDa beta chain. C5 is synthesized as asingle chain precursor protein product of a single copy gene (Havilandet al. (1991) J Immunol 146:362-368). The cDNA sequence of thetranscript of this gene predicts a secreted pro-C5 precursor of 1658amino acids along with an 18 amino acid leader sequence (see, e.g., U.S.Pat. No. 6,355,245).

The pro-C5 precursor is cleaved after amino acids 655 and 659, to yieldthe beta chain as an amino terminal fragment (amino acid residues+1 to655 of the above sequence) and the alpha chain as a carboxyl terminalfragment (amino acid residues 660 to 1658 of the above sequence), withfour amino acids (amino acid residues 656-659 of the above sequence)deleted between the two.

C5a is cleaved from the alpha chain of C5 by either alternative orclassical C5 convertase as an amino terminal fragment comprising thefirst 74 amino acids of the alpha chain (i.e., amino acid residues660-733 of the above sequence). Approximately 20 percent of the 11 kDamass of C5a is attributed to carbohydrate. The cleavage site forconvertase action is at, or immediately adjacent to, amino acid residue733 of the above sequence. A compound that would bind at, or adjacent,to this cleavage site would have the potential to block access of the C5convertase enzymes to the cleavage site and thereby act as a complementinhibitor.

C5 can also be activated by means other than C5 convertase activity.Limited trypsin digestion (see, e.g., Minta and Man (1997) J Immunol119:1597-1602 and Wetsel and Kolb (1982) J Immunol 128:2209-2216),thrombin, and acid treatment (Yamamoto and Gewurz (1978) J Immunol120:2008 and Damerau et al. (1989) Molec Immunol 26:1133-1142) can alsocleave C5 and produce active C5b.

Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotacticfactor, and C5b which through a series of protein interactions leads tothe formation of the lytic terminal complement complex, C5b-9. C5a andC5b-9 also have pleiotropic cell activating properties, by amplifyingthe release of downstream inflammatory factors, such as hydrolyticenzymes, reactive oxygen species, arachidonic acid metabolites andvarious cytokines.

C5b combines with C6, C7, and C8 to form the C5b-8 complex at thesurface of the target cell. Upon binding of several C9 molecules, themembrane attack complex (MAC, C5b-9, terminal complement complex—TCC) isformed. When sufficient numbers of MACs insert into target cellmembranes the openings they create (MAC pores) mediate rapid osmoticlysis of the target cells. Lower, non-lytic concentrations of MACs canproduce other effects. In particular, membrane insertion of smallnumbers of the C5b-9 complexes into endothelial cells and platelets cancause deleterious cell activation. In some cases activation may precedecell lysis.

As mentioned above, C3a and C5a are anaphylatoxins. These activatedcomplement components can trigger mast cell degranulation, whichreleases histamine from basophils and mast cells, and other mediators ofinflammation, resulting in smooth muscle contraction, increased vascularpermeability, leukocyte activation, and other inflammatory phenomenaincluding cellular proliferation resulting in hypercellularity. C5a alsofunctions as a chemotactic peptide that serves to attractpro-inflammatory granulocytes to the site of complement activation.

C5a receptors are found on the surfaces of bronchial and alveolarepithelial cells and bronchial smooth muscle cells. C5a receptors havealso been found on eosinophils, mast cells, monocytes, neutrophils, andactivated lymphocytes.

SUMMARY

The present disclosure relates to, inter alia, the generation by theinventors of a series of humanized monoclonal antibodies thatspecifically bind to free C5a protein (that is, C5a that has beenproteolytically cleaved from C5 protein), but not to paralog proteinfragments free C4a or free C3a [the antibodies are, often, referred toherein as anti-C5a antibodies or anti-C5a neoepitope antibodies]. Asdescribed herein and exemplified in the working examples, the generatedanti-C5a antibodies exhibit a high affinity for free C5a. For example,all of the humanized anti-C5a antibodies described herein bind to freeC5a with a K_(D) that is less than 1.25 nanomolar. Many of theantibodies bind to free C5a (e.g., free human C5a) with a K_(D) that isless than 300 picomolar; several of the antibodies bind to free C5a witha K_(D) that is less than 100 picomolar. In addition, the humanizedanti-C5a antibodies described herein also inhibit C5a-mediatedsignaling. Further structural and functional properties of theantibodies described herein are elaborated on below and exemplified inthe working examples.

The inventors have also demonstrated, using an animal model ofrheumatoid arthritis (RA) and a surrogate anti-mouse C5a antibody withproperties similar to the humanized antibody counterparts, efficacy ofanti-C5a antibodies in treating RA. Also shown in the working examplesare experiments demonstrating the positive therapeutic effects of ahumanized anti-C5a antibody in an animal model of human C5a-inducedneutropenia.

Accordingly, the inventors believe that the anti-C5a antibodies, orantigen-binding fragments thereof, described herein are useful in a hostof diagnostic and therapeutic methods related to disorders in whichC5a-mediated signaling contributes to pathogenesis. For example, theinventors assert that the humanized anti-C5a antibodies described hereinare useful for treating or preventing RA and other complement-associateddisorders including, but not limited to: atypical hemolytic uremicsyndrome (aHUS), age-related macular degeneration (AMD), sepsis, burn(e.g., severe burn), antiphospholipid syndrome (APS), acute respiratorydistress syndrome (ARDS), inflammation-related pain, asthma, lupusnephritis, intrauterine growth restriction (IUGR), HELLP syndrome(Hemolytic anemia, Elevated Liver enzymes and Low Platelet count),Goodpasture's syndrome, and chronic obstructive pulmonary disease(COPD). Additional disorders that are particularly amenable to treatmentwith a humanized anti-C5a antibody, or antigen-binding fragment thereof,are known in the art and recited herein.

The humanized anti-C5a antibodies described herein feature a number ofadvantages, e.g., over agents that bind to, and inhibit cleavage of,full-length or mature C5. Like such agents, the anti-C5a antibodies (andantigen-binding fragments thereof) described herein are capable ofinhibiting the anaphylatoxic downstream effects of C5 activation asmediated through C5 fragment C5a. That is, the anti-C5a antibodiesdescribed herein can inhibit the C5a-mediated inflammatory response,which is known to play an integral part in the pathogenesis ofcomplement-associated disorders such as, but not limited to, sepsis, RA,and asthma. However, as the concentration of C5 in human serum isapproximately 0.37 μM (Rawal and Pangburn (2001) J Immunol166(4):2635-2642), the use of high concentrations and/or frequentadministration of anti-C5 antibodies is often necessary to effectivelyinhibit C5, and thereby inhibit the C5a-mediated inflammatory response,in a human. Unlike C5, C5a is present in blood at much lowerconcentrations and is often restricted to specific areas of localcomplement activation such as, e.g., the lungs in asthma patients, thejoints of RA patients, or the drusen in the eyes of patients with AMD.Thus, the anti-C5a antibodies described herein can be administered(e.g., locally administered to sites of complement activation) to ahuman at a much lower dose and/or less frequently than, e.g., an anti-C5antibody, and effectively provide the same or greater inhibition of C5ain a human. The ability to administer a lower dose of the anti-C5aantibody, as compared to the required dose of an anti-C5 antibody, alsoallows for additional delivery routes such as, e.g., subcutaneousadministration, intramuscular administration, intrapulmonary delivery,and administration via the use of biologically degradable microspheres.A lower concentration of antigen C5a versus C5 also favors a longerhalf-life of the anti-C5a antibody, as compared to, e.g., the half-lifeof a therapeutic antibody that targets terminal complement, due to areduced contribution of antigen-mediated antibody clearance.

In addition, the anti-C5a antibodies described herein can also bedistinguished from therapeutic agents that inhibit terminal complement(such as C5 inhibitors) by their safety profile. A notable consequenceof inhibiting terminal complement components such as C5, C5b, C6, C7,C8, or C9 is decreased protection by the host immune system against theencapsulated bacteria that terminal complement ordinarily lyses—forexample, Neisseria meningitides and Neisseria gonorrhoeae. See, e.g.,Haeney et al. (1980) Clin Exp Immunol 40:16-24 and Brodsky (2009) Blood113(26):6522-6527. As the anti-C5a antibodies inhibit the C5a-mediatedinflammatory response, but do not prevent the formation of the terminalcomplement complex that lyses those encapsulated bacteria, patientsreceiving a therapeutic anti-C5a antibody described herein would notrequire a protective vaccination, e.g., a vaccination against Neisseriameningitides and Neisseria gonorrhoeae.

Accordingly, in one aspect, the disclosure features an isolatedantibody, or antigen-binding fragment thereof, that binds to free C5a.In some embodiments, the antibody or antigen-binding fragment thereofbinds to free human C5a (hC5a; e.g., a human C5a protein comprising, orconsisting of, the amino acid sequence depicted in SEQ ID NO:1). In someembodiments, the antibody can bind to a desarginated form of free C5a,e.g., the desarginated form of human C5a comprising, or consisting of,the amino acid sequence depicted in SEQ ID NO:2. The antibody can bindto a neoepitope of free C5a, which epitope is not present on uncleavedC5 or is present on only a minor fraction of total uncleaved C5.

While the disclosure is in no way limited to any particular theory ormechanism of action, in some embodiments, the anti-C5a antibody orantigen-binding fragment thereof binds to free C5a (e.g., free hC5a) andmay also bind to a subpopulation of uncleaved, processed C5 (e.g.,plasma C5) constituting less than 10 (e.g., less than 9.5, 9, 8.5, 8,7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3,0.2, or less than 0.1) % of the total population of full length C5 in asample (e.g., a blood or plasma sample or a sample comprisingrecombinant full length C5), which subpopulation is, in whole or inpart, denatured such that an otherwise occluded C5a neoepitope, to whichthe anti-C5a antibody or fragment binds, is exposed. Thus, an anti-C5aantibody or antigen-binding fragment thereof described herein can, insome embodiments, bind to free C5a, but not to the uncleaved C5 proteinof the 90% or greater uncleaved, native C5 population. In someembodiments, the above-described partially or fully denaturedsubpopulation of C5 is inactive or has reduced activity (e.g., less than90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5%of the activity of fully-functional, full-length C5 protein) in anynumber of suitable assays useful for testing C5 activity, e.g., ahemolytic assay or a CH50eq assay (see below). Suitable methods fortesting the activity of the minor subpopulation to which an anti-C5aantibody described herein may, in some embodiments, bind are known inthe art and described herein.

In some embodiments, any of the anti-C5a antibodies or antigen-bindingfragments thereof described herein do not inhibit C5 activity in an invitro hemolysis assay or an in vitro CH50eq assay even in the presenceof at least, equal to, or greater than a 5 (e.g., 5.6, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200)-fold excess of theanti-C5a antibody or antigen-binding fragment thereof to uncleaved C5(e.g., uncleaved, native C5). In some embodiments, any of the anti-C5aantibodies or antigen-binding fragments thereof described herein do notinhibit C5 activity in an in vitro hemolysis assay or an in vitro CH50eqassay even in the presence of between about a 5-fold to 200-fold (e.g.,between about 5-fold and 100-fold, between about 10-fold and 100-fold,between about 20-fold and 100-fold, or between about 10-fold and150-fold) excess of the anti-C5a antibody or antigen-binding fragmentthereof to uncleaved, native C5. Inhibition, e.g., as it pertains to C5activity, includes at least a 5 (e.g., at least a 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 60) % decrease in the activity ofuncleaved, native C5 in, e.g., a hemolytic assay or CH50eq assay ascompared to the effect of a control antibody (or antigen-bindingfragment thereof) under similar conditions and at an equimolarconcentration. Substantial inhibition, as used herein, refers toinhibition of a given activity (e.g., of C5 activity) of at least 40(e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 orgreater) %. In some embodiments, the C5 is obtained from plasma (e.g.,purified from or present in plasma, e.g., human plasma).

In some embodiments, the antibody or antigen-binding fragment thereofbinds to a C5a protein (e.g., a human C5a protein) with a K_(D) that isless than 2 nM. In some embodiments, the antibody or antigen-bindingfragment thereof binds to a C5a protein with a K_(D) that is less than 1nM [also referred to herein as “subnanomolar affinity”].

In some embodiments, the anti-C5a antibody or antigen-binding fragmentthereof binds to free C5a with a subnanomolar affinity [e.g., a K_(D) ofless than or equal to 9.9×10⁻¹⁰ (e.g., less than or equal to 9×10⁻¹⁰,8×10⁻¹⁰, 7×10⁻¹⁰, 6×10⁻¹⁰, 5×10⁻¹⁰, 4×10⁻¹⁰, 3×10⁻¹⁰, 2.5×10⁻¹⁰,2×10⁻¹⁰, 1×10⁻¹⁰, 8.0×10⁻¹¹, 7.0×10⁻¹¹, 6.0×10⁻¹¹, 5.0×10⁻¹¹, 4.0×10⁻¹¹,or 3.0×10⁻¹¹) M] in the presence of a molar excess of uncleaved, nativeC5 (e.g., purified and/or recombinant C5). In some embodiments, any ofthe anti-C5a antibodies or antigen-binding fragments thereof describedherein have at least a 100 (e.g., at least 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000)-foldgreater affinity (e.g., represented by its K_(D)) for free C5a than foruncleaved, native C5 protein.

Thus, in another aspect, the disclosure features an antibody orantigen-binding fragment thereof that (a) binds to free C5a (e.g., hC5a)with a subnanomolar affinity and (b) binds to free C5a with an affinitythat is at least 100 (e.g., at least 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000)-fold greaterthan its corresponding affinity for uncleaved, native C5 protein. Forexample, an anti-C5a antibody or antigen-binding fragment thereofdescribed herein can, in some embodiments, bind to free hC5a with aK_(D) of 100 nM and to at least a subpopulation of uncleaved human C5protein with a K_(D) that is at least 100-fold higher (e.g., at least 10nM).

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that binds to a free human C5apolypeptide having the amino acid sequence depicted in SEQ ID NO:1,wherein the antibody or antigen-binding fragment thereof binds to thehuman C5a polypeptide with a K_(D) that is less than 1.25×10⁻⁹ M in thepresence of a molar excess (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or500-fold molar excess) of uncleaved, native human C5 over human C5a(hC5a). In some embodiments, the antibody or antigen-binding fragmentthereof binds to a free hC5a polypeptide with a subnanomolar affinity(e.g., any of the subnanomolar K_(D)'s recited herein) in the presenceof at least, or greater than, a 2-fold molar excess, but no greater orless than a 500 (e.g., 500, 450, 400, 350, 300, 250, 200, 150, 100, 90,80, 70, 60, 50, 40, 30, 25, 20, or 15)-fold molar excess of uncleaved,native C5 over free hC5a. In some embodiments, the antibody orantigen-binding fragment thereof binds to a free hC5a polypeptide with asubnanomolar affinity (e.g., any of the subnanomolar K_(D)'s recitedherein) in the presence of between 2-fold and 20-fold molar excess ofuncleaved, native C5 over free hC5a. In some embodiments, an antibody orantigen-binding fragment thereof binds to a free hC5a polypeptide with asubnanomolar affinity (e.g., any of the subnanomolar K_(D)'s recitedherein) in the presence of between 10-fold and 20-fold molar excess ofuncleaved, native C5 over free hC5a. In some embodiments, an antibody orantigen-binding fragment thereof binds to a free hC5a polypeptide with asubnanomolar affinity (e.g., any of the subnanomolar K_(D)'s recitedherein) in the presence of between 5-fold and 15-fold molar excess ofuncleaved, native C5 over free hC5a. In some embodiments, an antibody orantigen-binding fragment thereof binds to a free hC5a polypeptide with asubnanomolar affinity (e.g., any of the subnanomolar K_(D)'s recitedherein) in the presence of at least 2-fold, but no greater than a20-fold molar excess of uncleaved, native C5 over free hC5a. Suchmeasurements can be in vitro measurements using, e.g., standard affinitydetermination techniques, many of which are recited and/or describedherein.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that binds to a human C5a polypeptidehaving the amino acid sequence depicted in SEQ ID NO:1, wherein theantibody or antigen-binding fragment thereof binds to the human C5apolypeptide with a K_(D) that is less than 1.25×10⁻⁹ M and wherein theantibody or antigen-binding fragment thereof does not substantiallyinhibit, as compared to an equimolar amount of a control antibody orantigen-binding fragment thereof, C5 activity even in the presence ofless than, or equal to, a 10-fold molar excess of the anti-C5a antibodyor antigen-binding fragment thereof to uncleaved, native C5.

In some embodiments of any of the anti-C5a antibodies or antigen-bindingfragments thereof described herein, the antibody or antigen-bindingfragment thereof binds to free human C5a and is cross-reactive with freeC5a from at least one non-human mammalian species. For example, in someembodiments, an anti-C5a antibody (or antigen-binding fragment thereof)binds to free C5a from human (e.g., with subnanomolar affinity) and alsobinds to free C5a from a non-human primate (e.g., cynomolgus macaque,rhesus macaque, ape, baboon, chimpanzee, orangutan, or gorilla), arodent (e.g., mouse, rat, hamster, Guinea pig, or rabbit), cow, goat,donkey, pig, dog, cat, or horse. In some embodiments, an anti-C5aantibody or antigen-binding fragment thereof described herein binds tofree hC5a with a K_(D) of less than or equal to 9.9×10⁻¹⁰ (e.g., lessthan or equal to 9×10⁻¹⁰, 8×10⁻¹⁰, 7×10⁻¹⁰, 6×10⁻¹⁰, 5×10⁻¹⁰, 4×10⁻¹⁰,3×10⁻¹⁰, 2.5×10⁻¹⁰, 2×10⁻¹⁰, 1×10⁻¹⁰, 8.0×10⁻¹¹, 7.0×10⁻¹¹, 6.0×10⁻¹¹,5.0×10⁻¹¹, 4.0×10⁻¹¹, or 3.0×10⁻¹¹) M and also binds to free C5a fromcynomolgus macaque (or another non-human primate species), wherein theaffinity (e.g., represented by its K_(D)) for human C5a is no more than500 (e.g., no more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125,150, 200, 250, 300, 350, 400, 450, or 475)-fold greater than theaffinity for cynomolgus macaque (or other non-human primate species)C5a. For example, in some embodiments, the anti-C5a antibody binds tofree hC5a with an affinity that is no more than 50-fold greater than thecorresponding affinity of the antibody for non-human primate C5a (e.g.,a K_(D) for free hC5a of 100 nM and a K_(D) for non-human primate C5a ofno more than 5 nM). In some embodiments, an anti-C5a antibody orantigen-binding fragment thereof described herein binds to free hC5awith a K_(D) of less than or equal to 9.9×10⁻¹⁰ (e.g., less than orequal to 9×10⁻¹⁰, 8×10⁻¹⁰, 7×10⁻¹⁰, 6×10⁻¹⁰, 5×10⁻¹⁰, 4×10⁻¹⁰, 3×10⁻¹⁰,2.5×10⁻¹⁰, 2×10⁻¹⁰, 1×10⁻¹⁰, 8.0×10⁻¹¹, 7.0×10⁻¹¹, 6.0×10⁻¹¹, 5.0×10⁻¹¹,4.0×10⁻¹¹ or 3.0×10⁻¹¹) M and also binds to C5a from a rodent (e.g.,mouse, rat, or rabbit), wherein the affinity (e.g., represented by itsK_(D)) for human C5a is no more than 1000 (e.g., no more than 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450,475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 975)-foldgreater than the affinity for rodent C5a. In some embodiments, any ofthe anti-C5a antibodies or antigen-binding fragments thereof describedherein bind with subnanomolar affinity to both human C5a and to C5a froma non-human mammal (e.g., a rodent or a non-human primate such ascynomolgus macaque). An antibody or antigen-binding fragment thereofcan, in some embodiments, bind to human C5a and non-human primate C5awith equal affinity (e.g., an equivalent K_(D)).

For example, the disclosure features an antibody or antigen-bindingfragment thereof that binds to free human C5a with subnanomolar affinity[e.g., a K_(D) of less than or equal to 9.9×10⁻¹⁰ (e.g., less than orequal to 9×10⁻¹⁰, 8×10⁻¹⁰, 7×10⁻¹⁰, 6×10⁻¹⁰, 5×10⁻¹⁰, 4×10⁻¹⁰, 3×10⁻¹⁰,2.5×10⁻¹⁰, 2×10⁻¹⁰, 1×10⁻¹⁰, 8.0×10⁻¹¹, 7.0×10⁻¹¹, 6.0×10⁻¹¹, 5.0×10⁻¹¹,4.0×10⁻¹¹, or 3.0×10⁻¹¹) M] and is cross-reactive with free C5a fromcynomolgus macaque (or other non-human primate), the antibody orantigen-binding fragment thereof binding to cynomolgus macaque (or othernon-human primate) C5a with a K_(D) of less than 10×10⁻⁹, 9×10⁻⁹,8×10⁻⁹, 7×10⁻⁹, 6×10⁻⁹, 5×10⁻⁹, 4×10⁻⁹, 3×10⁻⁹, 2×10⁻⁹, 1×10⁻⁹,9.9×10⁻¹⁰ (e.g., less than 9×10⁻¹⁰, 8×10⁻¹⁰, 7×10⁻¹⁰, 6×10⁻¹⁰, 5×10⁻¹⁰,4×10⁻¹⁰, 3×10⁻¹⁰, 2.5×10⁻¹⁰, 2×10⁻¹⁰, 1×10⁻¹⁰, or 8.0×10⁻¹¹) M], whereinthe affinity for human C5a is no more than 500 (e.g., no more than 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350,400, 450, or 475)-fold greater than the affinity for cynomolgus macaque(or non-human primate) C5a (e.g., K_(D) for human C5a of 100 nM and aK_(D) for non-human primate C5a of no more than 50 nM). Suitable methodsfor determining the affinity of an antibody or antigen-binding fragmentthereof for a given antigen are known in the art and described andexemplified herein.

In some embodiments, the cross-reactive anti-C5a antibody orantigen-binding fragment thereof functionally inhibits both free hC5aand the non-human mammalian C5a to which it binds. For example, anantibody inhibits by at least 70 (e.g., at least 75, 80, 85, 90, or 95or greater) % human C5a-dependent human neutrophil activation at a molarratio of 1:1 (antigen-binding site:C5a) and inhibits by at least 70(e.g., at least 75, 80, 85, 90, or 95 or greater) % non-human mammalianC5a-dependent neutrophil activation (the neutrophils being from the samespecies as the non-human mammalian C5a to which the antibody binds) at amolar ratio of 1:1 (antigen-binding site:C5a).

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that binds to a free hC5a polypeptidehaving the amino acid sequence depicted in SEQ ID NO:1, wherein theantibody or antigen-binding fragment thereof binds to the human C5apolypeptide with a K_(D) that is less than 1.25×10⁻⁹ M and wherein theantibody or antigen-binding fragment thereof binds to both hC5a and toC5a from a non-human mammalian species. The non-human mammalian speciescan be, e.g., a non-human primate such as cynomolgus macaque, rhesusmacaque, or baboon. In some embodiments, the non-human mammalian speciesis a rodent such as a mouse, rat, rabbit, Guinea pig, gerbil, orhamster. In some embodiments, the antibody or antigen-binding fragmentthereof binds to hC5a with an affinity no greater than 100-fold higherthan the corresponding affinity for C5a from the non-human mammalianspecies. In some embodiments, the antibody or antigen-binding fragmentinhibits by at least 50% human C5a-dependent human neutrophil activationat a molar ratio of 1:1 (antigen-binding site: C5a).

In some embodiments, the antibody or antigen-binding fragment thereofbinds to free C5a from a non-human primate (e.g., a cynomolgus macaqueor rhesus macaque), the free C5a protein having an amino acid sequencecomprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:179 or SEQ ID NO:180.

As described in the working examples, the inventors have also discovereda bivalent anti-C5a antibody, BNJ383, that binds to free C5a (in thiscase human C5a) with high affinity and, with a much lower affinity,uncleaved human C5 (hC5), wherein, in a composition (e.g., an aqueoussolution) under physiological conditions at equilibrium, and in thepresence of a molar excess of uncleaved human C5 as compared to themolar amount of the antigen-binding sites of the antibodies, at least95% of the plurality of antibodies each bind no more than one hC5molecule. The second antigen-binding site of the at least 95% of theplurality of antibodies remains available (e.g., substantiallyavailable) to bind to free C5a. While the disclosure is in no way boundby any particular theory or mechanism of action, the inventors believethat the bivalent anti-C5a antibody binds to uncleaved C5 in such a way(e.g., at such an epitope) that steric hindrance precludes, or at leastsubstantially inhibits, the binding of the second antigen-binding siteof the anti-C5a antibody to a second uncleaved C5 protein, although theantibody can easily accommodate the binding to two hC5a molecules. Thus,the antibody, even in a molar excess of uncleaved C5, retains theability to bind to free C5a with high affinity and thereby retains, evenin that molar excess, the ability to inhibit the pro-inflammatoryactivity of C5a.

One of ordinary skill in the art would easily and readily appreciate themyriad therapeutic benefits of such an anti-C5a antibody. For example,as noted above, the concentration of circulating C5 in human serum isvery high. Thus, when introduced into a mammal, an anti-C5a antibodythat is capable of simultaneously binding to two uncleaved C5 moleculeswould be rapidly inactivated in the molar excess of C5 and would then nolonger be capable of binding to free C5a in the event of complementactivation. And, as with anti-C5 antibodies, use of high concentrationsand/or frequent administration of this type of anti-C5a antibody wouldbe necessary to effectively inhibit C5a, in the event that it isproduced. In contrast, the anti-C5a antibody described herein thatretains the ability to bind to free C5a, even in a molar excess ofuncleaved C5, can thus be administered to a human at a much lower doseand/or less frequently than, e.g., an anti-C5 antibody and effectivelyprovide the same or greater inhibition of C5a in the human.

Accordingly, in yet another aspect, the disclosure features an isolatedantibody comprising two antigen-binding sites, wherein eachantigen-binding site independently can bind to free human C5a (hC5a) oruncleaved human C5 (hC5), wherein, in an aqueous solution comprising:(i) a plurality of said antibodies and (ii) a molar excess of hC5 ascompared to the molar amount of the antigen-binding sites, atequilibrium and under physiological conditions, at least 95 (e.g., atleast 95.5, 96, 96.5, 97, 97.5, or 97.7) % of said plurality ofantibodies bind no more than one hC5 molecule, i.e., no more than 5% ofthe antibodies are binding two molecules of hC5 at equilibrium.

In another aspect, the disclosure features an isolated antibodycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), wherein, at equilibrium and under physiological conditions, in anaqueous solution comprising: (i) a plurality of said antibodies and (ii)a molar excess of hC5 as compared to the molar amount of theantigen-binding sites (or antibodies), at least 95% of said plurality ofantibodies retain at least one antigen-binding site available to bindfree hC5a.

In another aspect, the disclosure features an isolated antibodycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), wherein, at equilibrium and under physiological conditions, in anaqueous solution comprising: (i) a plurality of said antibodies and (ii)a molar excess of hC5 as compared to the molar amount of theantigen-binding sites (or antibodies), each antigen-binding site of nomore than 5% of said plurality of antibodies is bound to a hC5 molecule.

In some embodiments of any of the isolated antibodies described herein,the molar excess is at least a two-fold (e.g., at least a 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or even 10-fold)molar excess.

In some embodiments of any of the isolated antibodies described herein,the physiological condition is 3.9 mM NaH₂PO₄, 6.1 mM Na₂HPO₄, and 150mM NaCl, at pH7.0.

In some embodiments of any of the isolated antibodies described herein,each antigen-binding site independently can bind to free hC5a with aK_(D) that is less than 1.25×10⁻⁹ M. In some embodiments, eachantigen-binding site can independently bind to free hC5a with asubnanomolar affinity (see above).

In some embodiments of any of the isolated antibodies described herein,the isolated antibody comprises a light chain polypeptide comprising theamino acid sequence depicted in SEQ ID NO:42 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:45.

In some embodiments of any of the isolated antibodies described herein,the isolated antibody comprises: (i) a light chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:20; (ii) a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:21; (iii) alight chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:22; (iv) a heavy chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:28; (v) a heavy chain CDR2 comprising the aminoacid sequence depicted in SEQ ID NO:46; and (vi) a heavy chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:47.

In some embodiments, the isolated antibody can comprise any of the lightchain CDR sets described herein, any of the light chain variable regionsdescribed herein (e.g., any of the humanized light chain variableregions), any of the heavy chain CDR sets described herein, any of theheavy chain variable regions described herein (e.g., any of thehumanized heavy chain variable regions), or any suitable combinationsthereof. See, e.g., Tables 1 or 2.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder), wherein the method includes administering to thehuman any of the isolated antibodies described herein in an amountsufficient to treat the complement-associated disorder.

In another aspect, the disclosure features a method for treating a humanafflicted with a C5a-associated complement disorder, wherein the methodcomprises administering to the human at least 0.6 (e.g., at least 0.7,0.8, 0.9, or 1) mg of any of the isolated antibodies described hereinper kg body weight of the human to thereby partially or completely bindand sequester nanogram levels of free C5a for greater than, equal to, orat least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) days.

In another aspect, the disclosure features a method for treating a humanafflicted with a C5a-associated complement disorder, wherein the methodcomprises administering to the human at least 10 mg of any of theisolated antibodies described herein per kg body weight of the human tothereby partially or completely bind and sequester nanogram levels offree C5a for at least 24 days.

In another aspect, the disclosure features a method for treating a humanafflicted with a C5a-associated complement disorder, wherein the methodcomprises administering to the human any of the isolated antibodiesdescribed herein (or for example a pharmaceutical composition comprisingany of the isolated antibodies described herein) in an amount sufficientto (a) achieve molar Cmax values equal to or less than the physiologicmolar concentration of uncleaved hC5 and (b) partially or completelybind and sequester pathophysiologic levels of free C5a.

In some embodiments of any of the methods described herein, an antibodyis administered to a subject (e.g., a human) in an amount sufficient toachieve a molar Cmax value that is substantially lower than thephysiologic molar concentration of uncleaved C5 (e.g., hC5).

In some embodiments of any of the methods described herein, the Cmaxvalue is, e.g., no greater than 80 nM (or approximately 0.6 mg/kg). Insome embodiments, Cmax levels are no greater than 70 (e.g., 60, 50, 40,30, or 20) nM. In some embodiments, the Cmax value is no greater thanapproximately 100 nM. In some embodiments, the Cmax value is no greaterthan 200 nM. In some embodiments of any of the methods described herein,the Cmax value is, e.g., no greater than 400 nM (or approximately 3mg/kg). In some embodiments, the Cmax value is no greater than 400 (e.g.350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10) nM.

In another aspect, the disclosure features a method for treating a humanafflicted with a C5a-associated complement disorder, wherein the methodcomprises administering to the human any of the isolated antibodiesdescribed herein (or for example a pharmaceutical composition comprisingany of the isolated antibodies described herein) in an amount sufficientto (a) achieve molar Cmax values equal to, less than, or substantiallylower than the molar physiologic concentration of uncleaved hC5 and (b)partially or completely bind and sequester nanogram levels of free C5afor at least 12 days (e.g., at least 24 days). Suitable Cmax values aredescribed above.

In some embodiments of any of the methods described herein, theC5a-associated complement disorder can be, e.g., one selected from thegroup consisting of sepsis, acute respiratory distress syndrome (ARDS),septic shock, anti-phospholipid syndrome, catastrophic anti-phospholipidsyndrome, disseminated intravascular coagulation, lupus nephritis,Goodpasture's Syndrome, burn or severe burn, asthma, HELLP syndrome(Hemolytic anemia, Elevated Liver enzymes and Low Platelet count),inflammation-induced pain, C5a-mediated neutropenia, age-related maculardegeneration (AMD), chronic obstructive pulmonary disease, andrheumatoid arthritis.

In yet another aspect, the disclosure features a composition comprisinga plurality of isolated antibodies, each antibody of the pluralitycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), and wherein, in the presence of human C5 (hC5) and underphysiological conditions, no more than 5% of the antibodies of theplurality at equilibrium comprise two antigen-binding sitessimultaneously bound to uncleaved hC5.

In some embodiments of any of the compositions described herein, thepercentage of the plurality in any particular binding configuration canbe evaluated using high performance liquid chromatography (HPLC). Insome embodiments, the physiological conditions in which the antibodiesare evaluated comprises the following conditions: incubation of hC5(e.g., a molar excess (e.g., a 2-fold molar excess) of hC5) with theplurality of antibodies at 4° C. for 84 hours in an aqueous solutioncomprising 3.9 mM NaH₂PO₄, 6.1 mM Na₂HPO₄, and 150 mM NaCl, at pH7.0.For the purposes of this disclosure, the solution obtained at 84 hoursat 4° C. is considered to be at equilibrium.

In some embodiments of any of the compositions described herein, no morethan 5% of the antibodies of the plurality comprise two antigen-bindingsites simultaneously bound to uncleaved hC5 under physiologicalconditions and in the presence of at least a 2-fold molar excess of hC5to antibody.

In some embodiments of any of the compositions described herein, no morethan 5% of the antibodies of the plurality comprise two antigen-bindingsites simultaneously bound to uncleaved hC5 molecules as evaluated usingHPLC following incubation of the plurality of antibodies with hC5 at 4°C. for 84 hours in an aqueous solution comprising 3.9 mM NaH₂PO₄, 6.1 mMNa₂HPO₄, and 150 mM NaCl, at pH7.0.

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, each antibody of the pluralitycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), and wherein no more than 5% of the antibodies of the pluralitycomprise two antigen-binding sites simultaneously bound to uncleaved hC5as evaluated (e.g., using HPLC) following incubation of the plurality ofantibodies with hC5 at 4° C. for 84 hours in an aqueous solutioncomprising 3.9 mM NaH₂PO₄, 6.1 mM Na₂HPO₄, and 150 mM NaCl, at pH7.0.

In yet another aspect, the disclosure features a composition comprisinga plurality of isolated antibodies, each antibody of the pluralitycomprising a first antigen-binding site and a second antigen-bindingsite, wherein each antigen-binding site independently can bind to freehuman C5a (hC5a) or uncleaved human C5 (hC5), wherein eachantigen-binding site independently can bind to the free hC5a polypeptidewith a K_(D) that is less than 1.25×10⁻⁹ M, and wherein, in the presenceof human C5 (hC5) and as evaluated (e.g., using high performance liquidchromatography (HPLC)) under physiological conditions, the twoantigen-binding sites of at least 95% of the plurality of antibodies areoccupied by uncleaved hC5 in the following configurations: (i) the firstantigen-binding site binds uncleaved hC5 and the second antigen-bindingsite is unbound; or (ii) the first antigen-binding site is unbound andthe second antigen-binding site binds uncleaved hC5.

In yet another aspect, the disclosure features a composition comprisinga plurality of isolated antibodies, each antibody of the pluralitycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), and wherein, in the presence of human C5 (hC5) and as evaluated(e.g., using high performance liquid chromatography (HPLC)) underphysiological conditions, at least 95% of the antibodies of theplurality comprise at least one antigen-binding site capable of bindingto free hC5a.

In some embodiments of any of the compositions described herein, theplurality of antibodies is evaluated in the presence of at least a2-fold molar excess of hC5:antibody. In some embodiments of any of thecompositions described herein, the plurality of antibodies is evaluatedin the presence of at least a 2-fold molar excess of hC5:antigen-bindingsites.

In yet another aspect, the disclosure features an isolated antibodycomprising two antigen-binding sites, wherein the antibody binds to freeC5a or uncleaved C5, and wherein one of the antigen-binding sites of theantibody remains available to bind free C5a in the presence of a molarexcess (e.g., at least or greater than a 2-fold, 5-fold, 10-fold,15-fold, or even a 20-fold molar excess) of uncleaved C5.

In some embodiments, the antigen-binding sites have the same specificity(e.g., the CDRs of each of the two antigen-binding sites share identicalamino acid sequences). In some embodiments, free C5a is human C5a. Insome embodiments, the antibody is cross-reactive between human C5a andC5a from a non-human mammalian species. The antibody can, in someembodiments, bind to free C5a with a subnanomolar affinity. In someembodiments, the antibody has an affinity for C5a that is at least100-fold greater than its corresponding affinity for uncleaved C5.

In another aspect, the disclosure features an isolated antibodycomprising two antigen-binding sites, wherein each antigen-binding siteindependently binds to free human C5a (hC5a) or uncleaved human C5(hC5), and wherein, at any concentration of uncleaved hC5 (e.g., in amolar excess of uncleaved hC5 over hC5a), at least one of theantigen-binding sites of the antibody remains available to bind to freehC5a (e.g., under human physiological conditions, e.g., in human bloodor serum).

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), and wherein, at a molar ratio of 1:1 (antibody:hC5), no morethan, or less than, 5 (e.g., no more than, or less than 4.9, 4.8, 4.7,4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3,3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9,1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1) % of the antibodies of theplurality comprise two antigen-binding sites simultaneously bound touncleaved hC5. In some embodiments, each antigen-binding siteindependently can bind to free hC5a with a K_(D) that is less than1.25×10⁻⁹ M (or, for example, with subnanomolar affinity).

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), and wherein, in the presence of physiologic levels ofuncleaved hC5, the antibodies of the plurality partially or completelybind and sequester nanogram levels of free C5a for greater than, equalto, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25) days when administered to a human at doses of 1 mg/kg orhigher.

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), and wherein, in the presence of physiologic levels ofuncleaved hC5, the antibodies of the plurality partially or completelybind and sequester nanogram levels of free C5a for greater than, equalto, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25) days when administered to a human at doses of 10 mg/kg orhigher.

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), and wherein, in the presence of physiologic levels ofuncleaved hC5, the antibodies of the plurality partially or completelybinds and sequesters nanogram levels of free C5a for greater than, equalto, or at least 12 (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25) days when administered at doses achieving molar Cmax valuessubstantially lower than the molar physiologic concentration ofuncleaved hC5.

In another aspect, the disclosure features a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), and wherein, in the presence of physiologic levels ofuncleaved hC5, the antibodies of the plurality partially or completelybind and sequester pathophysiologic levels of free C5a when administeredat doses achieving molar Cmax values substantially lower than the molarphysiologic concentration of un-cleaved hC5.

In another aspect, the disclosure features an isolated antibodycomprising a first antigen-binding site and a second antigen-bindingsite, wherein each antigen-binding site independently can bind to freehuman C5a (hC5a) or uncleaved human C5 (hC5), and wherein, when bothantigen-binding sites are fully-occupied (and, e.g., under humanphysiological conditions, e.g., in human blood or serum), the followingbinding configurations are possible: (i) the first antigen-binding sitebinds free hC5a and the second antigen-binding site binds uncleaved hC5;(ii) the first antigen-binding site binds free hC5a and the secondantigen-binding site binds free hC5a; or (iii) the first antigen-bindingsite binds uncleaved hC5 and the second antigen-binding site binds freehC5a.

In yet another aspect, the disclosure features an isolated antibodycomprising a first antigen-binding site and a second antigen-bindingsite, wherein each antigen-binding site independently can bind to freehuman C5a (hC5a) or uncleaved human C5 (hC5), wherein eachantigen-binding site independently can bind to free hC5a with a K_(D)that is less than 1.25×10⁻⁹ M (or, for example, with subnanomolaraffinity), and wherein, in a physiological solution containing aplurality of the antibodies, at least 95% of the antibodies are in thefollowing configurations: (i) the first antigen-binding site binds freehC5a and the second antigen-binding site binds uncleaved hC5; (ii) thefirst antigen-binding site binds free hC5a and the secondantigen-binding site binds free hC5a; (iii) the first antigen-bindingsite binds uncleaved hC5 and the second antigen-binding site binds freehC5a; (iv) the first antigen-binding site binds uncleaved hC5 and thesecond antigen-binding site is unbound; (v) the first antigen-bindingsite binds hC5a and the second antigen-binding site is unbound; (vi) thefirst antigen-binding site is unbound and the second antigen-bindingsite binds uncleaved hC5; (vii) the first antigen-binding site isunbound and the second antigen-binding site binds hC5a; and (viii) thefirst antigen-binding site is unbound and the second antigen-bindingsite is unbound.

In another aspect, the disclosure features an isolated antibodycomprising two antigen-binding sites, wherein each antigen-binding siteindependently can bind to free human C5a (hC5a) or uncleaved human C5(hC5), and wherein, in a molar excess of uncleaved hC5 over hC5a, theantibody inhibits by at least 50% hC5a-dependent human neutrophilactivation at a molar ratio of 1:1 (antigen-binding site: hC5a).

In some embodiments of any of the antibodies described herein, theconfigurations are possible under human physiological conditions withfully-folded, native, human C5a and C5 proteins.

In some embodiments of any of the antibodies described herein, theantibody binds to free hC5a with a K_(D) that is less than 1.25×10⁻⁹ M(or, for example, with subnanomolar affinity).

In yet another aspect, the disclosure features an antibody that (a)binds to free C5a (e.g., hC5a) with a subnanomolar affinity and (b)binds to free C5a with an affinity that is at least 100 (e.g., at least110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, or 10000)-fold greater than its corresponding affinity foruncleaved C5 protein. In a physiologic composition comprising aplurality of the antibodies, for at least 95% of the antibodies, onlyone antigen-binding site of the antibody binds to uncleaved C5 protein,whereas the second antigen-binding site remains available to bind tofree C5a. (The hC5a can have the amino acid sequence depicted in SEQ IDNO:1.)

In another aspect, the disclosure features a method for treating a humanafflicted with a C5a-associated complement disorder, the methodcomprising administering to the human a composition comprising aplurality of isolated antibodies, wherein each antibody of the pluralitycomprises two antigen-binding sites, wherein each of the antigen-bindingsites independently can bind to free human C5a (hC5a) or uncleaved humanC5 (hC5), wherein at least 1 mg of the antibodies per kg body weight ofthe human is administered to the human, and wherein administration ofthe antibodies is effective to partially or completely bind andsequester nanogram levels of free C5a for at least 12 days.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder), the method comprising administering to the human acomposition comprising a plurality of isolated antibodies, wherein eachantibody of the plurality comprises two antigen-binding sites, whereineach of the antigen-binding sites independently can bind to free humanC5a (hC5a) or uncleaved human C5 (hC5), wherein at least 10 mg of theantibodies per kg body weight of the human is administered to the human,and wherein administration of the antibodies is effective to partiallyor completely bind and sequester nanogram levels of free C5a for atleast 24 days.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder), the method comprising administering to the human acomposition comprising a plurality of isolated antibodies, wherein eachantibody of the plurality comprises two antigen-binding sites, whereineach of the antigen-binding sites independently can bind to free humanC5a (hC5a) or uncleaved human C5 (hC5), wherein the antibodies areadministered at a dose sufficient to: (a) achieve molar Cmax valuessubstantially lower than the molar physiologic concentration ofuncleaved hC5 and (b) partially or completely bind and sequesternanogram levels of free C5a for at least 12 days.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder), the method comprising administering to the human acomposition comprising a plurality of isolated antibodies, wherein eachantibody of the plurality comprises two antigen-binding sites, whereineach of the antigen-binding sites independently can bind to free humanC5a (hC5a) or uncleaved human C5 (hC5), wherein the antibodies areadministered at a dose sufficient to: (a) achieve molar Cmax valuessubstantially lower than the molar physiologic concentration ofuncleaved hC5 and (b) partially or completely bind and sequesternanogram levels of free C5a for at least 24 days.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder), the method comprising administering to the human acomposition comprising a plurality of isolated antibodies, wherein eachantibody of the plurality comprises two antigen-binding sites, whereineach of the antigen-binding sites independently can bind to free humanC5a (hC5a) or uncleaved human C5 (hC5), wherein the antibodies areadministered at a dose sufficient to: (a) achieve molar Cmax valuessubstantially lower than the molar physiologic concentration ofuncleaved hC5 and (b) partially or completely bind and sequesterpathophysiologic levels of free C5a.

It is understood that any of the compositions (e.g., comprising aplurality of antibodies) or isolated antibodies (e.g., that retain, inthe presence of C5 or molar excess of C5, a free Fab arm capable ofbinding to free C5a) described herein can be: (a) formulated aspharmaceutical compositions in accordance with the disclosure, (b)included in therapeutic kits (described herein), or (c) included in thepre-filled syringes described herein.

As described in the working examples provided herein, the inventors havealso discovered an antibody, BNJ383 (see below), that not only bindswith high affinity (subnanomolar affinity) to free hC5a, but atconcentrations in excess of uncleaved C5 also inhibits terminalcomplement complex (TCC) formation in a dose dependent manner. Even atconcentrations of the anti-C5a antibody in greater than 6.5-fold excessof C5, however, inhibition of TCC is not complete. While the disclosureis by no means limited by any particular theory or mechanism of action,the antibody may inhibit TCC formation by binding to at least a fractionof uncleaved C5 and preventing its cleavage and/or otherwise preventingthe successful association of C5 with additional TCC components. Theinventors appreciated that such an antibody is useful for treatingcomplement-associated disorders, e.g., in which C5a plays a significantrole and the C5b-containing TCC may play a less substantial role. Suchdisorders can include, e.g., sepsis, acute respiratory distress syndrome(ARDS), septic shock, anti-phospholipid syndrome, catastrophicanti-phospholipid syndrome, disseminated intravascular coagulation,lupus nephritis, Goodpasture's Syndrome, burn or severe burn, asthma,HELLP syndrome (Hemolytic anemia, Elevated Liver enzymes and LowPlatelet count), inflammation-induced pain, C5a-mediated neutropenia,age-related macular degeneration (AMD), chronic obstructive pulmonarydisease, and rheumatoid arthritis.

The inventors also appreciated that use of such an anti-C5a antibody totreat these conditions, among others, may provide an even morebeneficial safety profile as compared to use of terminal complementinhibitory drugs. As noted above, one notable consequence of inhibitingterminal complement components such as C5, C5b, C6, C7, C8, or C9 isdecreased protection by the host immune system against the encapsulatedbacteria that terminal complement ordinarily lyses—for example,Neisseria meningitides and Neisseria gonorrhoeae. As the anti-C5aantibodies described in this section inhibit the C5a-mediatedinflammatory response, but do not completely inhibit the formation ofthe terminal complement complex that lyses those encapsulated bacteria,patients receiving a therapeutic anti-C5a antibody described herein maynot require a protective vaccination, e.g., a vaccination againstNeisseria meningitides and Neisseria gonorrhoeae. Partial inhibition ofthe TCC, while not wholly abrogating terminal complement'santi-microbial response, may in fact reduce TCC-induced inflammation astissue injury. The partial TCC inhibition, in combination withinhibition of C5a, is believed to make the anti-C5a antibody an evenmore potent anti-inflammatory compound.

Accordingly, in another aspect, the disclosure features an antibody orantigen-binding fragment thereof that binds to free C5a, wherein thefree C5a is human C5a having the amino acid sequence depicted in SEQ IDNO:1, wherein the antibody inhibits the binding of C5a to C5a receptor,and wherein the antibody partially inhibits formation of the terminalcomplement complex (TCC). Partial inhibition by an anti-C5a antibody orantigen-binding fragment thereof described herein can be, e.g., acomplement activity that is, in the presence of the antibody, up to, orno greater than, 80 (e.g., 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or25) % of the complement activity in the absence of the antibody orantigen-binding fragment thereof. In some embodiments, the antibody orantigen-binding fragment thereof binds to free C5a (e.g., free hC5a)with a subnanomolar affinity. In some embodiments, the antibody orantigen-binding fragment thereof has an affinity for free C5a that is atleast 100-fold greater than the corresponding affinity of the antibodyor antigen-binding fragment for uncleaved C5. In some embodiments, theantibody or antigen-binding fragment thereof inhibits by at least 50%formation of TCC at concentrations exceeding 200 (e.g., 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, or 400 or more)μg/mL as measured using a CH50eq assay. In some embodiments, theantibody or antigen-binding fragment thereof inhibits by at least 50%classical complement pathway activation at concentrations exceeding 200(e.g., 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360,380, or 400 or more) μg/mL as measured using the Wieslab® ClassicalPathway Complement Kit as described in the working examples.

In another aspect, the disclosure features a method for treating a humanafflicted with a complement-associated disorder (e.g., a C5a-associatedcomplement disorder or a complement-associated inflammatory disorder).The method includes administering to the human an effective amount of anantibody or antigen-binding fragment thereof that inhibits the bindingof C5a to C5a receptor, and wherein the antibody partially inhibitsformation of the terminal complement complex (TCC). See above. Thedisorder can be any of those known in the art or described herein.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that binds to a human C5a polypeptidehaving the amino acid sequence depicted in SEQ ID NO:1, but does notbind to the alpha chain of uncleaved, native C5, wherein the antibody orantigen-binding fragment thereof binds to the human C5a polypeptide witha K_(D) that is less than 1.25×10⁻⁹ M.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that binds to a human C5a polypeptidehaving the amino acid sequence depicted in SEQ ID NO:1, but does notbind to the alpha chain of uncleaved, native C5, wherein the antibodyinhibits by at least 50% human C5a-dependent human neutrophil activationat a molar ratio of 1:1 (antigen-binding site:C5a). In some embodiments,the antibody inhibits by at least 50% human C5a-dependent humanneutrophil migration in an assay in which 0.4 nM of antibody is used toinhibit the neutrophil-activation activity of 2 nM human C5a asdescribed in Example 5. In some embodiments, the antibody does notcomprise exemplary CDR pairing 3 depicted in Table 1. In someembodiments, the antibody is not BNJ371.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to a human C5a polypeptide having theamino acid sequence depicted in SEQ ID NO:2.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprising:a light chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:20; a light chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:21; and a light chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:22.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprising:a light chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:20; a light chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:38; and a light chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:22.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprising:a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:28; a heavy chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:29; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:30.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprising:a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:28; a heavy chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:67; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:30.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprising:a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:28; a heavy chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:46; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:47.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:37 or SEQ ID NO:36.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:27 or SEQ ID NO:33.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:37 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:27.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:36 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:33.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:19 or SEQ ID NO:17.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:27 or SEQ ID NO:25.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:19 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:27.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:17 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:25.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:42 or SEQ ID NO:40.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:27 or SEQ ID NO:33.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:42 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:27.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:40 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:33.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:17 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:33.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in any one of SEQ ID NO:45, SEQ IDNO:44, or SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:19 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:45.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:17 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:44.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:17 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:37 or SEQ ID NO:36.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:45 or SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:37 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:45.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:36 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:42 or SEQ ID NO:40.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heavy chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:45 or SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:42 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:45.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a light chain polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO:40 and a heavy chainpolypeptide comprising the amino acid sequence depicted in SEQ ID NO:49.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than7×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than5×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than3×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than2.5×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than1.5×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than1.0×10⁻¹⁰ M.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein binds to hC5a with a K_(D) that is less than8.0×10⁻¹¹ M.

In some embodiments, an antibody inhibits by at least 70 (e.g., at least75, 80, 85, 90, or 95 or greater) % human C5a-dependent human neutrophilactivation at a molar ratio of 1:1 (antigen-binding site:C5a). In someembodiments, the antibody does not comprise exemplary CDR pairing 3depicted in Table 1. In some embodiments, the antibody is not BNJ371.

In yet another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that comprises a light chain CDR set asset forth in Table 3 or Table 7. For example, the isolated antibody orantigen-binding fragment thereof can comprise a light chain polypeptidecomprising: (i) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:140; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:96; and a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:142; (ii) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:156; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:157;and a light chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:158; (iii) a light chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:164; a light chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:165; and a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:166; (iv) alight chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:172; a light chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:173; and a light chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:174; (v) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:84; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:85; and a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:86;(vi) a light chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:92; a light chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:89; and a light chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:93; (vii) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:88; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:89;and a light chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:90; (viii) a light chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:95; a light chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:96; and a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:97; (ix) alight chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:99; a light chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:100; and a light chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:101; (x) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:84; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:85; and a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:103; (xi) alight chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:105; a light chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:106; and a light chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:107; (xii) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:92; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:89; and a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:108;(xiii) a light chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:20; a light chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:110; and a light chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:111; or (xiv) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:20; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:21;and a light chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:113. In some embodiments, the antibody or antigen-bindingfragment thereof comprising the light chain CDR set also comprises aheavy chain polypeptide comprising any one of the heavy chain CDR setsas set forth in Table 8.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that comprises a heavy chain CDR set asset forth in Table 3 or Table 8. For example, in some embodiments anisolated antibody or antigen-binding fragment thereof described hereincomprises a heavy chain polypeptide comprising: (i) a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:115; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:144;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:117; (ii) a heavy chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:28; a heavy chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:67; and a heavy chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:30; (iii) aheavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:160; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:161; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:162; (iv) a heavy chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:168; a heavy chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:169; and aheavy chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:170; (v) a heavy chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:176; a heavy chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:177; and a heavy chain CDR3 comprisingthe amino acid sequence depicted in SEQ ID NO:178; (vi) a heavy chainCDR1 comprising the amino acid sequence depicted in SEQ ID NO:115; aheavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:116; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:117; (vii) a heavy chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:119; a heavy chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:120; and a heavy chainCDR3 comprising the amino acid sequence depicted in SEQ ID NO:121;(viii) a heavy chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:115; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:123; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:117; (ix) a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:115; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:124;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:117; (x) a heavy chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:119; a heavy chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:126; and a heavy chain CDR3 comprisingthe amino acid sequence depicted in SEQ ID NO:127; (xi) a heavy chainCDR1 comprising the amino acid sequence depicted in SEQ ID NO:115; aheavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:129; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:117; (xii) a heavy chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:131; a heavy chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:132; and a heavy chainCDR3 comprising the amino acid sequence depicted in SEQ ID NO:133;(xiii) a heavy chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:28; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:46; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:47; or (xiv) a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:136; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:137;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:138. In some embodiments, the antibody or antigen-bindingfragment thereof comprising the heavy chain CDR set also comprises alight chain polypeptide comprising any one of the light chain CDR setsas set forth in Table 7.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof comprising a light chain CDR set fromTable 7 and its cognate heavy chain CDR set as set forth in Table 8. Inanother aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof comprising a paired light chain andheavy chain CDR set as set forth in Table 3 or Table 9. For example, insome embodiments an isolated antibody, or antigen-binding fragmentthereof, described herein comprises: (i) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:140; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:96; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:142;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:144; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (ii) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:20; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:21; and a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:22;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:28; a heavy chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:67; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:30; (iii) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:156; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:157; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:158; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:160;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:161; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:162; (iv) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:164; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:165; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:166; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:168;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:169; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:170; (v) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:172; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:173; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:174; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:176;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:177; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:178; (vi) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:88; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:89; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:90; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:119;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:120; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:121; (vii) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:105; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:106; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:107; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:115;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:124; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:117; (viii) a light chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:84; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:85; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:86;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:116; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (ix) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:20; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:110; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:111;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:136; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:137; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:138; (x) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:20; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:21; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:113;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:28; a heavy chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:46; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:47; (xi) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:99; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:100; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:101; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:119;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:126; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:127; (xii) a light chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:95; a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:96; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:97; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:115;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:144; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:117; (xiii) a light chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:140; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:96; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:142;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:123; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (xiv) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:105; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:106; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:107;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:123; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (xv) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:92; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:89; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:108;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:144; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (xvi) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:92; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:89; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:93;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:123; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (xvii) a light chain CDR1 comprisingthe amino acid sequence depicted in SEQ ID NO:92; a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:89; a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:93;a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:115; a heavy chain CDR2 comprising the amino acid sequence depictedin SEQ ID NO:144; and a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:117; (xviii) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:84; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:85;a light chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:103; a heavy chain CDR1 comprising the amino acid sequence depictedin SEQ ID NO:115; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:129; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:117; (xix) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:95; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:96;a light chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:97; a heavy chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:115; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:123; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:117; (xx) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:84; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:85;a light chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:103; a heavy chain CDR1 comprising the amino acid sequence depictedin SEQ ID NO:115; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:144; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:117; or (xxi) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:20; a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:21;a light chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:113; a heavy chain CDR1 comprising the amino acid sequence depictedin SEQ ID NO:131; a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:132; and a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:133.

In some embodiments, an antibody or antigen-binding fragment thereofcomprises a paired light chain CDR set and heavy chain CDR set as setforth in Table 3. In some embodiments, an antibody or antigen-bindingfragment thereof comprises a paired light chain CDR set and heavy chainCDR set as set forth in Table 2. For example, the disclosure features anantibody comprising: (i) a light chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:20; (ii) a light chain CDR2 comprisingthe amino acid sequence depicted in SEQ ID NO:21; and (iii) a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:22;(iv) a heavy chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:28; (v) a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:46; and (vi) a heavy chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:47.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises a light chain variable region set forth in Table 2, whichlight chain is paired with any one of the heavy chain variable regionsset forth in Table 2. For example, the disclosure features an antibody(or an antigen-binding fragment thereof) comprising: (a) a light chainvariable region having an amino acid sequence comprising the amino acidsequence depicted in SEQ ID NO:42 and (b) a heavy chain variable regionhaving an amino acid sequence comprising the amino acid sequencedepicted in SEQ ID NO:45.

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein comprises: (i) a heavy chain variable region frameworkregion 1 comprising the amino acid sequence depicted in SEQ ID NO:68 orSEQ ID NO:69; (ii) a heavy chain variable region framework region 2comprising the amino acid sequence depicted in SEQ ID NO:70 or SEQ IDNO:71; and a heavy chain variable region framework region 3 comprisingthe amino acid sequence depicted in any one of SEQ ID NOs:72 to 74. Insome embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region framework region 4 comprisingthe amino acid sequence depicted in SEQ ID NO:75. In some embodiments,the antibody or antigen-binding fragment thereof comprises a heavy chainvariable region comprising the amino acid sequence depicted in any oneof SEQ ID NOs:76 to 80. The antibody heavy chain can comprise any of theheavy chain CDR sets described herein. The heavy chain variable regioncan be, in some embodiments, paired with the variable region polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:16.

In some embodiments, an antibody or antigen-binding fragment thereofbinds to a non-human C5a protein. For example, the antibody orantigen-binding fragment thereof can bind to mouse C5a and/ordesarginated mouse C5a protein. In some embodiments, an isolatedantibody or antigen-binding fragment thereof can bind to mouse C5a(and/or desarginated mouse C5a) and comprise: (i) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:54; (ii) alight chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:55; (iii) a light chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:56; (iv) a heavy chain CDR1 comprising the aminoacid sequence depicted in SEQ ID NO:62; (v) a heavy chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:63; and (vi) aheavy chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:64. In some embodiments, the anti-mouse C5a antibody can comprise alight chain polypeptide comprising the amino acid sequence depicted inSEQ ID NO:59; and a heavy chain polypeptide comprising the amino acidsequence depicted in SEQ ID NO:66.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein inhibits the interaction between C5a and a C5areceptor. The C5a receptor can be, e.g., C5aR1 or C5L2.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein does not substantially inhibitcomplement-mediated hemolysis of red blood cells in vitro and/or invivo.

In some embodiments, an isolated antibody (and accordingly anyantigen-binding fragment thereof) is a monoclonal antibody, a humanizedantibody, or a fully-human antibody.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein is selected from the group consisting of arecombinant antibody, a single chain antibody, a diabody, an intrabody,a chimerized or chimeric antibody, a deimmunized human antibody, an Fvfragment, an Fd fragment, an Fab fragment, an Fab′ fragment, and anF(ab′)₂ fragment.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein is multispecific (e.g., bispecific) in that theantibody or fragment binds to at least two different epitopes. The twodifferent epitopes can be, e.g., two different epitopes from the sameprotein (e.g., C5a) or the antibody can bind to a first epitope from afirst protein (e.g., C5a) and a second epitope from a second protein.

In some embodiments, an isolated antibody or antigen-binding fragmentthereof described herein comprises a heterologous moiety. Theheterologous moiety can be, e.g., a sugar. For example, the antibody orantigen-binding fragment thereof can be glycosylated. The heterologousmoiety can be, e.g., a detectable label such as, but not limited to, afluorescent label, a luminescent label, a heavy metal label, aradioactive label, or an enzymatic label.

In some embodiments, an isolated anti-C5a antibody or antigen-bindingfragment thereof described herein is modified with a moiety thatimproves the stabilization and/or retention of the antibody incirculation. For example, the modification can be PEGylation orhesylation. In another embodiment, the anti-C5a antibody can contain analtered constant region that has reduced (or no) effector function, ascompared to the effector function of its corresponding unalteredconstant region. In some embodiments, the anti-C5a antibody contains analtered constant region that has between about 0 to about 20% of theeffector function of the unaltered constant region. Exemplaryembodiments of such decreased-effector function antibodies are describedherein.

In another aspect, the disclosure features an isolated antibody orantigen-binding fragment thereof that crossblocks the binding of any oneof the foregoing antibodies.

In yet another aspect, the disclosure features a pharmaceuticalcomposition comprising one or more of any of the isolated antibodies orantigen-binding fragments thereof described herein and apharmaceutically-acceptable carrier, diluent, and/or excipient.

In another aspect, the disclosure features: (i) a nucleic acid encodingone or more of any of the antibodies or antigen-binding fragmentsthereof described herein; (ii) a vector comprising the nucleic acid;(iii) an expression vector comprising the nucleic acid; and/or (iv) acell comprising the vector or the expression vector. In another aspect,the disclosure features a method for producing a polypeptide (such asany of the antibodies or antigen-binding fragments thereof describedherein). The method comprises culturing the aforementioned cell(comprising the expression vector) under conditions and for a timesufficient to allow expression by the cell of the antibody orantigen-binding fragment encoded by the nucleic acid in the vector. Themethod can also include isolated the antibody or antigen-bindingfragment from the cell or from the medium in which the cell is cultured.

In another aspect, the disclosure features an isolated nucleic acidencoding any of the amino acid sequences described herein or apolypeptide having an amino acid sequence comprising, or consisting of,any of the amino acid sequences set forth herein. The nucleic acid canbe included in a vector, e.g., an expression vector, and/or can bepresent in a cell.

In yet another aspect, the disclosure features a therapeutic kitcomprising: (i) one or more of the isolated antibodies orantigen-binding fragments described herein (e.g., one or more of any ofthe humanized antibodies or antigen-binding fragments thereof describedherein); and (ii) means for delivery of the antibody or antigen-bindingfragment to a subject. The means can be suitable for, e.g., subcutaneousdelivery, intraocular delivery, or intraarticular delivery of theantibody or antigen-binding fragment thereof to the subject. The meanscan be, e.g., a syringe, a double-barreled syringe, or two separatesyringes incorporated for use in administering a therapeutic antibody orantigen-binding fragment thereof, while drawing off knee fluid (e.g.,for analysis) in a push-pull fashion. In some embodiments, the means isfor ocular delivery and comprises a trans-scleral patch or a contactlens, each of which comprises the antibody or antigen-binding fragmentthereof. In some embodiments, the means is suitable for intrapulmonarydelivery. For example, the means can be an inhaler or a nebulizer. Insome embodiments, the means is a pre-filled syringe such as a pendevice. The pre-filled syringe can contain, e.g., at least onepharmaceutical unit dosage form of one or more of the antibodies orantigen-binding fragments thereof provided herein.

In some embodiments, the therapeutic kits described herein can containat least one additional active agent for use in treating acomplement-associated disorder in a subject. The additional active agentcan be, e.g., any of the additional agents described herein.

In yet another aspect, the disclosure features a method for treating orpreventing a complement-associated disorder. The method includesadministering to a human in need thereof a therapeutic antibody orantigen-binding fragment thereof described herein in an amountsufficient to treat a complement-associated disorder afflicting thehuman. The method can also include identifying the subject as having acomplement-associated disorder. The complement-associated disorder canbe, e.g., a complement-associated inflammatory disorder, atypicalhemolytic uremic syndrome, age-related macular degeneration, rheumatoidarthritis, sepsis, or antiphospholipid syndrome. In some embodiments,the complement-associated disorder is a complement-associated pulmonarydisorder. For example, the complement-associated pulmonary disorder canbe, e.g., asthma or chronic obstructive pulmonary disease. Othercomplement-associated disorders amenable to treatment or prevention asset forth in the method are described herein. The mode ofadministration, which can vary depending on the type ofcomplement-associated disorder to be treated, can be, e.g., intravenousadministration, intrapulmonary administration, intraocularadministration, subcutaneous administration, or intraarticularadministration.

In some embodiments, the antibody or antigen-binding fragment thereof isadministered to the human in an amount and with a frequency sufficientto maintain a reduced level of systemic C5a activity for the duration ofthe treatment. In some embodiments, the methods can include after theadministering, monitoring the human for an improvement in one or moresymptoms of the complement-associated disorder.

In some embodiments, the methods can include administering to the humanone or more additional therapeutic agents.

In yet another aspect, the disclosure features an article ofmanufacture, which comprises: (i) a container with a label and (ii) acomposition comprising an antibody or antigen-binding fragment thereofdescribed herein. The label can indicate that the composition is to beadministered to a human having, suspected of having, or at risk fordeveloping, a complement-associated disorder. The article of manufacturecan also include one or more additional active agents.

As used throughout the present disclosure, the term “antibody” refers toa whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) moleculethat is generated by any one of a variety of methods that are known inthe art and described herein. The term “antibody” includes a polyclonalantibody, a monoclonal antibody, a chimerized or chimeric antibody, ahumanized antibody, a deimmunized human antibody, and a fully humanantibody. The antibody can be made in or derived from any of a varietyof species, e.g., mammals such as humans, non-human primates (e.g.,monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats,dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Theantibody can be a purified or a recombinant antibody.

As used herein, the term “antibody fragment,” “antigen-bindingfragment,” or similar terms refer to a fragment of an antibody thatretains the ability to bind to an antigen (e.g., an epitope present inC5a, but not in the alpha chain of uncleaved, native C5 protein), e.g.,a single chain antibody (scFv), an Fd fragment, an Fab fragment, an Fab′fragment, or an F(ab′)₂ fragment. An scFv is a single polypeptide chainthat includes both the heavy and light chain variable regions of theantibody from which the scFv is derived. In addition, diabodies (Poljak(1994) Structure 2(12):1121-1123; Hudson et al. (1999) J Immunol Methods23(1-2):177-189, the disclosures of both of which are incorporatedherein by reference in their entirety), minibodies, triabodies(Schoonooghe et al. (2009) BMC Biotechnol 9:70), domain antibodies (alsoknown as “heavy chain immunoglobulins” or camelids; Holt et al. (2003)Trends Biotechnol 21(11):484-490); and intrabodies (Huston et al. (2001)Hum Antibodies 10(3-4):127-142; Wheeler et al. (2003) Mol Ther8(3):355-366; Stocks (2004) Drug Discov Today 9(22): 960-966, thedisclosures of each of which are incorporated herein by reference intheir entirety) are included in the definition of antibody fragments andcan be incorporated into the compositions, and used in the methods,described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating complement-associated disorders in a subject, will beapparent from the following description, the examples, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Venn diagram depicting the degree of overlap of the epitopesin human C5a bound by a select set of murine anti-human C5a antibodies:5an101ME, 5an180ME, 5an048ME, 5an179ME, and 5an178ME.

FIG. 2 is a line graph depicting the antagonism of C5a-mediatingsignaling in vitro using a neutrophil activation assay. The Y-axisrepresents the optical density (OD) measurement of a chromogenicsubstrate as a function of myeloperoxidase release by freshly isolatedhuman neutrophils. The X-axis represents the concentration of antibodyincubated with the cells. The humanized antibodies tested—BNJ367,BNJ369, BNJ371, BNJ378, BNJ381, BNJ383, and a humanizedanti-C5antibody—are identified in the inset legend.

FIG. 3 is a line graph depicting the effect of several therapeuticantibodies on joint inflammation in a mouse model of rheumatoidarthritis. The Y-axis represents the thickness of the initially inflamedknee joint in millimeters. The X-axis represents the days after diseaseonset. The therapeutic antibodies tested—5an195ME (a mouse anti-mouseC5a antibody) and a control antibody with the same Fc region as theanti-C5a antibody—are identified in the inset legend.

FIG. 4 is a line graph depicting the effect of several therapeuticantibodies on overall disease severity in a mouse model of rheumatoidarthritis. The Y-axis represents the arthritis index. The X-axisrepresents the days after disease onset. The therapeutic antibodiestested—5an195ME (a mouse anti-mouse C5a antibody) and a control antibodywith the same Fc region as the anti-C5a antibody—are identified in theinset legend.

FIG. 5 sets forth a series of humanized heavy chain variable regionsequences. In order from uppermost to lowermost: the heavy chainvariable region of the BNJ345 humanized anti-C5a antibody (SEQ IDNO:76); the heavy chain variable region of the BNJ346 humanized anti-C5aantibody (SEQ ID NO:77); the heavy chain variable region of the BNJ347humanized anti-C5a antibody (SEQ ID NO:78); the heavy chain variableregion of the BNJ354 humanized anti-C5a antibody (SEQ ID NO:79); and theheavy chain variable region of the BNJ350 humanized anti-C5a antibody(SEQ ID NO:80). The skilled artisan will appreciate the delineationbetween heavy chain framework regions 1, 2, 3, and 4 and the heavy chainCDRs 1, 2, and 3. Such delineations as defined by Kabat et al. (infra)are shown in the figure. “HC FR1” refers to heavy chain variable regionframework region 1, “HC FR2” refers to heavy chain variable regionframework region 2, “HC FR3” refers to heavy chain variable regionframework region 3, and “HC FR4” refers to heavy chain variable regionframework region 4. “HC CDR1” refers to heavy chain variable regioncomplementarity determining region (CDR) 1, “HC CDR2” refers to heavychain variable region CDR2, and “HC CDR3” refers to heavy chain variableregion CDR3.

FIG. 6 is a line graph depicting the percentage of circulatingneutrophils in the blood of mice following administration of hC5a to themice. On the Y-axis, neutrophil counts are expressed as a percentage of“baseline,” which is the neutrophil count at time 0 (or 100%neutrophils). The X-axis represents time in minutes. Mouse cohorts wereintravenously administered a control antibody [anti-anthrax protectiveantigen 63, IgG2/G4 isotype] (“control”; five mice) or the anti-humanC5a antibody BNJ383 at one of the following doses: 24 mg/kg (five mice);12 mg/kg (five mice); 6 mg/kg (five mice); and 3 mg/kg (five mice) andthen later were administrated hC5a. See Example 13. Six mice, “sham,”were not administered human C5a.

FIG. 7 is a bar graph depicting the myeloperoxidase (MPO) level in theplasma of mice before and following administration of human C5a to themice. The Y-axis represents the concentration (ng/mL) of MPO in mouseplasma. The X-axis represents time in minutes. Mouse cohorts wereintravenously administered a control antibody [anti-anthrax protectiveantigen 63, IgG2/G4 isotype] (“control”; eight mice) or the anti-humanC5a antibody BNJ383 at one of the following doses: 24 mg/kg (five mice);12 mg/kg (five mice); 6 mg/kg (five mice); and 3 mg/kg (five mice) andthen later were administrated hC5a. Four mice, “sham,” were notadministered human C5a.

FIG. 8 is a line graph depicting the change in human C5a level in plasmaof mice (administered human C5a) in the presence or absence of differentconcentrations of an anti-hC5a antibody (BNJ383). The Y-axis representsthe concentration (ng/mL) of hC5a in mouse plasma. The X-axis representstime in minutes. Mouse cohorts were intravenously administered a controlantibody [anti-anthrax protective antigen 63, IgG2/G4 isotype](“control”; six mice) or the anti-human C5a antibody BNJ383 at one ofthe following doses: 24 mg/kg (three mice); 12 mg/kg (three mice); 6mg/kg (three mice); and 3 mg/kg (three mice) and then later wereadministrated hC5a. Four mice, “sham,” were not administered human C5a.

FIG. 9 is a line graph depicting the competition for binding to humanC5a in vitro. 250 pM of ruthenium-labeled anti-C5a antibody (BNJ383) wasincubated with 1 nM biotinylated hC5a, along with various concentrations(e.g., 400, 133, 44.4, 14.8, 4.9, 1.6, and 0.5 nM) of one of thefollowing: (a) human C5a desarg protein in phosphate-buffered saline,(b) human plasma, (c) cynomolgus macaque plasma, (d) Balb/C (mouse)plasma, or (e) DBA/2J (mouse) plasma. With respect to the plasmacomponents (b), (c), (d), and (e), the concentration refers to theapproximate final concentration of C5 antigen in the incubation mixture.The Y-axis represents arbitrary fluorescence units as a function of theamount of ruthenium-labeled anti-C5a antibody detected. The X-axisrepresents concentration (nM) of the antigen competitor.

FIG. 10 is a line graph depicting the effect of several complementinhibitory proteins on the alternative pathway (AP) of complement. TheY-axis represents the percentage of AP complement activity as comparedto baseline (BL; the level of activity in the absence of a complementinhibitor). The X-axis represents the concentration of a givencomplement inhibitor (μM). The effects of the anti-hC5a antibody,BNJ383, along with an anti-C5 antibody on AP activity were eachevaluated.

FIG. 11 is a line graph depicting the effect of several complementinhibitory proteins on the classical pathway (CP) of complement. TheY-axis represents the percentage of CP complement activity as comparedto baseline (BL; the level of activity in the absence of a complementinhibitor). The X-axis represents the concentration of a givencomplement inhibitor (μM). The effects of the anti-hC5a antibody,BNJ383, along with an anti-C5 antibody on CP activity were eachevaluated.

FIGS. 12A, 12B, 12C, and 12D are a series of chromatographs depictingthe retention times of the anti-C5a antibody (BNJ383) and an anti-hC5antibody in the presence or absence of hC5 protein. For all of thepanels, the X-axis represents retention time in minutes and the Y-axisrepresents the absorbance units at 214 nm wavelength. In each panel, theinlayed subpanel depicts an enhanced view of the featured peaks.

FIG. 12A depicts the retention time for BNJ383 in the absence of hC5protein.

FIG. 12B depicts the retention time for the anti-C5 antibody in theabsence of hC5 protein.

FIG. 12C depicts the retention time for BNJ383 in the presence of hC5(2.1-fold molar excess of hC5 over BNJ383). From right to left, theenumerated peaks represent: (a) uncomplexed BNJ383 or hC5; (b) BNJ383with one antigen-binding site bound to uncleaved hC5; and (c) a minorfraction consistent with dual occupancy of BNJ383 with uncleaved hC5.

FIG. 12D depicts the retention time for the anti-C5 antibody in thepresence of an equimolar amount of hC5. From right to left, theenumerated peaks represent: (a) uncomplexed anti-C5 antibody or hC5; (b)the anti-C5 antibody with one antigen-binding site bound to uncleavedhC5; and (c) the anti-C5 antibody bound to two uncleaved C5 molecules.

FIG. 13 is a line graph depicting the binding of the anti-C5a antibodyBNJ383 to hC5a desarg in the presence of hC5 using an ELISA. The X-axisrepresents the concentration (ng/mL) of the antibody. The Y-axisrepresents the optical density at 450 nm wavelength.

FIG. 14 is a scatter plot depicting the concentration of free C5a/C5adesarg present in the plasma of cynomolgus macaques as a function of theplasma concentration of anti-C5a antibody (BNJ383). The Y-axis depictsthe concentration (pg/mL) of C5a/C5a desarg detected in plasma samplesfrom cynomolgus macaques at time points ranging from 1 day to 30 daysfollowing a single dose intravenous administration of BNJ383 at 1 mg/kg,10 mg/kg, 100 mg/kg, 250 mg/kg, or 400 mg/kg body weight of the animals.The X-axis represents the concentration of the BNJ383 (μg/mL) in each ofthe samples.

FIG. 15A is a scatter plot depicting the percentage of hemolyticactivity in serum samples relative to baseline values (Y-axis) initiatedvia the classical pathway as a function of the concentration of anti-C5aantibody (BNJ383) in circulation (X-axis).

FIG. 15B is a scatter plot depicting the percentage of terminalcomplement complex formation initiated via the classical pathway inserum samples measured by a CH50eq assay relative to baseline values(Y-axis) as a function of the concentration of anti-C5a antibody(BNJ383) in circulation (X-axis).

FIG. 15C is a scatter plot depicting the percentage of terminalcomplement complex formation initiated via the classical pathway inserum samples measured by a CCP assay relative to baseline values(Y-axis) as a function of the concentration of anti-C5a antibody(BNJ383) in circulation (X-axis).

DETAILED DESCRIPTION

The present disclosure provides antibodies and antigen-binding fragmentsthereof that bind to free C5a (e.g., free human C5a), compositionscontaining the antibodies or their fragments, and methods for using anyof the foregoing to treat or prevent complement-associated disorderssuch as, but not limited to, aHUS, macular degeneration (e.g., AMD), RA,sepsis, antiphospholipid syndrome, burn (e.g., severe burn),Goodpasture's syndrome, lupus nephritis, or a complement-associatedpulmonary disorder such as asthma or chronic obstructive pulmonarydisease (COPD). The disclosure also provides anti-C5a antibodies (andfragments thereof) that are cross-reactive between free C5a from humanand free C5a from a non-human mammalian species such as a non-humanprimate (e.g., cynomolgus macaque or rhesus macaque). While in no wayintended to be limiting, exemplary antibodies (and antigen-bindingfragments), compositions (e.g., pharmaceutical compositions andformulations), and methods for using the compositions are elaborated onbelow and exemplified in the working Examples.

Anti-C5a Antibodies and Antigen-Binding Fragments Thereof

The disclosure provides antibodies that bind to complement componentC5a. As discussed above, the proform of C5, a 1676 amino acid residueprecursor protein, is processed by a series of proteolytic cleavageevents. The first 18 peptides (numbered −18 to −1) constitute a signalpeptide that is cleaved from the precursor protein. The remaining 1658amino acid protein is cleaved in two places to form the alpha and betachains. The first cleavage event occurs between amino acid residues 655and 656. The second cleavage occurs between amino acid residues 659 and660. The two cleavage events result in the formation of three distinctpolypeptide fragments: (i) a fragment comprising amino acids 1 to 655,which is referred to as the beta chain; (ii) a fragment comprising aminoacids 660 to 1658, which is referred to as the alpha chain; and (iii) atetrapeptide fragment consisting of amino acids 656 to 659. The alphachain and the beta chain polypeptide fragments are connected to eachother via disulfide bond and constitute the mature C5 protein. The CP orAP C5 convertase activates mature C5 by cleaving the alpha chain betweenresidues 733 and 734, which results in the liberation of C5a fragment(amino acids 660 to 733). The remaining portion of mature C5 is fragmentC5b, which contains the residues 734 to 1658 of the alpha chaindisulfide bonded to the beta chain.

In vivo, C5a is rapidly metabolized by a serum enzyme, carboxypeptidaseB, to a 73 amino acid form termed “C5a desarg,” which has lost thecarboxyterminal arginine residue. Accordingly, in some embodiments, anantibody that binds to free C5a also binds to desarginated C5a. In someembodiments, an antibody that binds to C5a does not bind to desarginatedC5a.

In some embodiments, the anti-C5a antibody binds to a neoepitope presentin C5a, i.e., an epitope that becomes exposed upon the liberation of C5afrom the alpha chain fragment of mature C5. That is, in someembodiments, an anti-C5a antibody described herein binds to C5a and/orC5a desarg, but not to uncleaved, native (fully-folded) C5.

As described above, in some embodiments, the anti-C5a antibody orantigen-binding fragment thereof can bind to a subpopulation ofuncleaved, processed C5 (e.g., plasma C5) constituting less than 10(e.g., less than 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, or less than 0.1) % of the totalpopulation of full length C5 in a sample (e.g., a blood or plasma sampleor a sample comprising recombinant full length C5), which subpopulationis in whole, or in part, denatured such that an otherwise occluded C5aneoepitope is exposed. Thus, an anti-C5a antibody or antigen-bindingfragment thereof described herein can, in some embodiments, bind to freeC5a, but not to C5 of the 90% or greater uncleaved, native C5population. In some embodiments, the partially or fully denaturedsubpopulation is inactive or has reduced activity (e.g., less than 90,85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5% ofthe activity of fully-functional, full-length C5 protein) in any numberof suitable assays useful for testing C5 activity, e.g., a hemolyticassay or a CH50eq assay. Suitable methods for testing the activity ofthe minor subpopulation to which an anti-C5a antibody described hereinmay, in some embodiments, bind are known in the art and describedherein.

In some embodiments, the anti-C5a antibody binds to a mammalian (e.g.,human) C5a protein. For example, the anti-C5a antibody can bind to ahuman C5a protein having the following amino acid sequence:TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGR (SEQ ID NO:1). In some embodiments, an anti-C5aantibody can bind to a desarginated human C5a protein having thefollowing amino acid sequence:TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLG (SEQ ID NO:2). An anti-C5a antibody describedherein can bind to both full-length human C5a and desarginated humanC5a.

In some embodiments, the antibody can bind to human C5a at an epitopewithin or overlapping with a structural fragment of the protein havingthe amino acid sequence: TLQKKIEEIAAKYK (SEQ ID NO:3); HSVVKKCCYDGAC(SEQ ID NO:4); VNNDE (SEQ ID NO:5); TCEQRAAR (SEQ ID NO:6); ISLG (SEQ IDNO:7); PRCIKAFTECCVVASQLRANIS (SEQ ID NO:8); HKDMQLG (SEQ ID NO:9); orHKDMQLGR (SEQ ID NO:10). See, e.g., Cook et al. (2010) Acta CrystD66:190-197. In some embodiments, the antibody can bind to C5a at anepitope within or overlapping with the amino acid sequence of a peptidefragment of C5a comprising at least two of the paired cysteine residues.For example, an anti-C5a antibody can bind to fragment comprising, orconsisting of, the amino acid sequence: CCYDGACVNNDETC (SEQ ID NO:11);CYDGACVNNDETCEQRAARISLGPRCIKAFTEC (SEQ ID NO:12; andCEQRAARISLGPRCIKAFTECC (SEQ ID NO:13), wherein, in each of the peptidefragments, the first and final cysteine residues are paired by disulfidebonds. In some embodiments, an anti-C5a antibody described herein canbind to a human C5a protein at an epitope within, or overlapping with,the amino acid sequence: YDGACVNNDETCEQRAAR (SEQ ID NO:14) orCYDGACVNNDETCEQRAA (SEQ ID NO:15). In some embodiments, an antibody canbind to a human C5a protein or fragment thereof containing an amino acidsequence that contains, or consists of, at least four (e.g., at leastfour, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17or more) consecutive amino acids depicted in any one of SEQ ID NOs:1 to15. In some embodiments, an anti-C5a antibody described herein binds toa ternary epitope comprising two or more (e.g., at least two, three, orfour) discontinuous peptide regions of C5a protein, e.g., two or morediscontinuous C5a peptide regions joined together by way of a disulfidelinkage.

Methods for identifying the epitope to which a particular antibody(e.g., an anti-C5a antibody) binds are also known in the art. Forexample, the binding epitope within C5a (or desarginated C5a) to whichan anti-C5a antibody binds can be identified by measuring the binding ofthe antibody to several (e.g., three, four, five, six, seven, eight,nine, 10, 15, 20, or 30 or more) overlapping peptide fragments of acomplement component C5a protein (e.g., several overlapping fragments ofa human C5a protein having the amino acid sequence depicted in SEQ IDNO:1 or SEQ ID NO:2). Each of the different overlapping peptides is thenbound to a unique address on a solid support, e.g., separate wells of amulti-well assay plate. Next, the anti-C5a antibody is interrogated bycontacting it to each of the peptides in the assay plate for an amountof time and under conditions that allow for the antibody to bind to itsepitope. Unbound anti-C5a antibody is removed by washing each of thewells. Next, a detectably-labeled secondary antibody that binds to theanti-C5a antibody, if present in a well of the plate, is contacted toeach of the wells, and unbound secondary antibody is removed by washingsteps. The presence or amount of the detectable signal produced by thedetectably-labeled secondary antibody in a well is an indication thatthe anti-C5a antibody binds to the particular peptide fragmentassociated with the well. See, e.g., Harlow and Lane (supra), Benny K.C. Lo (supra), and U.S. Patent Application Publication No. 20060153836,the disclosure of which is incorporated by reference in its entirety. Aparticular epitope to which an antibody binds can also be identifiedusing BIAcore chromatographic techniques (see, e.g., PharmaciaBIAtechnology Handbook, “Epitope Mapping,” Section 6.3.2 (May 1994); andJohne et al. (1993) J Immunol Methods 160:20191-8).

In some embodiments, an anti-C5a antibody described herein contains aspecific set of light chain complementarity determining regions (CDRs)and/or a specific set of heavy chain CDRs. For example, in someembodiments an anti-C5a antibody or antigen-binding fragment thereofdescribed herein can comprise a light chain CDR set obtained from alight chain polypeptide comprising the amino acid sequence depicted inany one of SEQ ID NOs:19, 37, or 42. In some embodiments, an anti-C5aantibody or antigen-binding fragment thereof described herein cancomprise a heavy chain CDR set obtained from a heavy chain polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:27 or 45.Exemplary light chain and heavy chain CDR sets obtained from theaforementioned light chain variable regions and heavy chain variableregions are described below in more detail (see Table 1).

The exact boundaries of CDRs, and framework regions, have been defineddifferently according to different methods. In some embodiments, thepositions of the CDRs or framework regions within a light or heavy chainvariable domain can be as defined by Kabat et al. [(1991) “Sequences ofProteins of Immunological Interest.” NIH Publication No. 91-3242, U.S.Department of Health and Human Services, Bethesda, Md.]. In such cases,the CDRs can be referred to as “Kabat CDRs” (e.g., “Kabat LCDR2” or“Kabat HCDR1”). In some embodiments, the positions of the CDRs of alight or heavy chain variable region can be as defined by Chothia et al.(1989) Nature 342:877-883. Accordingly, these regions can be referred toas “Chothia CDRs” (e.g., “Chothia LCDR2” or “Chothia HCDR3”). In someembodiments, the positions of the CDRs of the light and heavy chainvariable regions can be as defined by a Kabat-Chothia combineddefinition. In such embodiments, these regions can be referred to as“combined Kabat-Chothia CDRs.” In some embodiments, the positions of theCDRs and/or framework regions within a light or heavy chain variabledomain can be as defined by Honnegger and Plückthun (2001) J Mol Biol309: 657-670. Identification of the CDRs within a light chain or heavychain variable region using the aforementioned definitions is well knownin the art of antibody engineering. For example, Thomas et al. [(1996)Mol Immunol 33(17/18):1389-1401] exemplifies the identification of lightchain and heavy chain CDR boundaries according to Kabat and Chothiadefinitions.

Accordingly, in some embodiments an anti-C5a antibody or antigen-bindingfragment thereof described herein can comprise a Kabat-defined, aChothia-defined, or a combined Kabat-Chothia-defined light chain CDR setobtained from a light chain polypeptide comprising the amino acidsequence depicted in any one of SEQ ID NOs:19, 37, or 42. In someembodiments, an anti-C5a antibody or antigen-binding fragment thereofdescribed herein can comprise a Kabat-defined, a Chothia-defined, or acombined Kabat-Chothia-defined heavy chain CDR set obtained from a heavychain polypeptide comprising the amino acid sequence depicted in SEQ IDNO:27 or 45.

In some embodiments, an anti-C5a antibody described herein comprises alight chain variable region containing one or more of: a light chainCDR1 comprising or consisting of the following amino acid sequence:RASESVDSYGNSFMH (SEQ ID NO:20); a light chain CDR2 comprising orconsisting of the following amino acid sequence: RASNLES (SEQ ID NO:21);and a light chain CDR3 comprising or consisting of the following aminoacid sequence: QQSNEDPYT (SEQ ID NO:22). In some embodiments, ananti-C5a antibody described herein comprises a light chain variableregion containing each of a light chain CDR1 comprising or consisting ofthe following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO:20); alight chain CDR2 comprising or consisting of the following amino acidsequence: RASNLES (SEQ ID NO:21); and a light chain CDR3 comprising orconsisting of the following amino acid sequence: QQSNEDPYT (SEQ IDNO:22). Exemplary anti-C5a antibodies comprising such a light chainvariable domain include, e.g., the BNJ364, BNJ367, BNJ378, BNJ366,BNJ369, and BNJ383 anti-C5a antibodies described herein (vide infra;Table 2).

In some embodiments, an anti-C5a antibody described herein comprises alight chain variable region containing one or more of: a light chainCDR1 comprising or consisting of the following amino acid sequence:RASESVDSYGNSFMH (SEQ ID NO:20); a light chain CDR2 comprising orconsisting of the following amino acid sequence: WASTRES (SEQ ID NO:38);and a light chain CDR3 comprising or consisting of the following aminoacid sequence: QQSNEDPYT (SEQ ID NO:22). In some embodiments, ananti-C5a antibody described herein comprises a light chain variableregion containing each of a light chain CDR1 comprising or consisting ofthe following amino acid sequence: RASESVDSYGNSFMH (SEQ ID NO:20); alight chain CDR2 comprising or consisting of the following amino acidsequence: WASTRES (SEQ ID NO:38); and a light chain CDR3 comprising orconsisting of the following amino acid sequence: QQSNEDPYT (SEQ IDNO:22). Exemplary anti-C5a antibodies comprising such a light chainvariable domain include, e.g., the BNJ371 and BNJ381 anti-C5a antibodiesdescribed herein (vide infra; Table 2).

In some embodiments, an anti-C5a antibody described herein comprises aheavy chain variable region containing one or more of: a heavy chainCDR1 comprising or consisting of the following amino acid sequence:DYSMD (SEQ ID NO:28); a heavy chain CDR2 comprising or consisting of thefollowing amino acid sequence: AINPNSGGTNYNQKFKD (SEQ ID NO:29); and aheavy chain CDR3 comprising or consisting of the following amino acidsequence: SGSYDGYYAMDY (SEQ ID NO:30). In some embodiments, an anti-C5aantibody described herein comprises a heavy chain variable regioncontaining each of a heavy chain CDR1 comprising or consisting of thefollowing amino acid sequence: DYSMD (SEQ ID NO:28); a heavy chain CDR2comprising or consisting of the following amino acid sequence:AINPNSGGTNYNQKFKD (SEQ ID NO:29); and a heavy chain CDR3 comprising orconsisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ IDNO:30). Exemplary anti-C5a antibodies comprising such a heavy chainvariable domain include, e.g., the BNJ364, BNJ367, BNJ371, and BNJ378anti-C5a antibodies described herein (vide infra; Table 2).

In some embodiments, an anti-C5a antibody described herein comprises aheavy chain variable region containing one or more of: a heavy chainCDR1 comprising or consisting of the following amino acid sequence:DYSMD (SEQ ID NO:28); a heavy chain CDR2 comprising or consisting of thefollowing amino acid sequence: AIHLNTGYTNYNQKFKG (SEQ ID NO:46); and aheavy chain CDR3 comprising or consisting of the following amino acidsequence: GFYDGYSPMDY (SEQ ID NO:47). In some embodiments, an anti-C5aantibody described herein comprises a heavy chain variable regioncontaining each of a heavy chain CDR1 comprising or consisting of thefollowing amino acid sequence: DYSMD (SEQ ID NO:28); a heavy chain CDR2comprising or consisting of the following amino acid sequence:AIHLNTGYTNYNQKFKG (SEQ ID NO:46); and a heavy chain CDR3 comprising orconsisting of the following amino acid sequence: GFYDGYSPMDY (SEQ IDNO:47). Exemplary anti-C5a antibodies comprising such a heavy chainvariable domain include, e.g., the BNJ366, BNJ369, BNJ381, and BNJ383anti-C5a antibodies described herein (vide infra; Table 2).

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof described herein can contain a heavy chain CDR2 regioncomprising the amino acid sequence: AINPNSGGTNYSQKFKD (SEQ ID NO:67).For example, an anti-C5a antibody described herein can comprise a heavychain variable region containing one or more of: a heavy chain CDR1comprising or consisting of the following amino acid sequence: DYSMD(SEQ ID NO:28); a heavy chain CDR2 comprising or consisting of thefollowing amino acid sequence: AINPNSGGTNYSQKFKD (SEQ ID NO:67); and aheavy chain CDR3 comprising or consisting of the following amino acidsequence: SGSYDGYYAMDY (SEQ ID NO:30). In some embodiments, an anti-C5aantibody described herein comprises a heavy chain variable regioncontaining each of a heavy chain CDR1 comprising or consisting of thefollowing amino acid sequence: DYSMD (SEQ ID NO:28); a heavy chain CDR2comprising or consisting of the following amino acid sequence:AINPNSGGTNYSQKFKD (SEQ ID NO:67); and a heavy chain CDR3 comprising orconsisting of the following amino acid sequence: SGSYDGYYAMDY (SEQ IDNO:30). An example of an anti-C5a antibody described herein, whichcontains such a heavy chain polypeptide and binds to human C5a with aK_(D) that is less than 1 nM is the 5an101ME antibody described below.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof described herein contains one of the exemplary light chain CDRset and heavy chain CDR set pairings 1 to 4 depicted in Table 1.

TABLE 1 Exemplary Heavy and Light Chain CDR Pairings Exemplary ExemplaryLight Chain Heavy Chain Antibodies with CDR Pairings CDR1 CDR2 CDR3 CDR1CDR2 CDR3 such Pairings 1 SIN: 20 SIN: 21 SIN: 22 SIN: 28 SIN: 29 SIN:30 BNJ364, BNJ367, BNJ378 2 SIN: 20 SIN: 21 SIN: 22 SIN: 28 SIN: 46 SIN:47 BNJ366, BNJ369, BNJ383 3 SIN: 20 SIN: 38 SIN: 22 SIN: 28 SIN: 29 SIN:30 BNJ371 4 SIN: 20 SIN: 38 SIN: 22 SIN: 28 SIN: 46 SIN: 47 BNJ381 “SIN”refers to “SEQ ID NO.”The amino acid sequences represented by the SEQ ID NOs in Table 1 areset forth in Table 2.

In some embodiments, the anti-C5a antibody does not comprise exemplaryCDR pairing 3. In some embodiments, the anti-C5a antibody is not BNJ371.

In some embodiments, the light chain polypeptide of an anti-C5a antibodydescribed herein can be a λ light chain polypeptide (e.g., a fully humanor humanized λ light chain polypeptide). In some embodiments, the lightchain polypeptide of an anti-C5a antibody described herein is a κ lightchain polypeptide (e.g., a fully human or humanized κ light chainpolypeptide). The amino acid sequences of numerous light chainpolypeptides (e.g., numerous human light chain polypeptides) arewell-known in the art and set forth in, e.g., Kabat et al. (1991),supra. Exemplary κ light chain polypeptide amino acid sequences are setforth in Table 2.

In some embodiments, an anti-C5a antibody described herein can comprisea light chain constant region. For example, the light chain constantregion can be a λ light chain polypeptide constant region or a κ lightchain constant region. The amino acid sequence for a number of human λand κ light chain constant regions are known in the art and describedin, e.g., Kabat et al. (1991), supra. Exemplary κ light chainpolypeptide amino acid sequences are set forth in Table 2.

The heavy chain polypeptide can comprise a constant region (e.g., aheavy chain constant region 1 (CH1), heavy chain constant region 2(CH2), heavy chain constant region 3 (CH3), a heavy chain constantregion 4 (CH4), or a combination of any of the foregoing). The heavychain polypeptide can comprise an Fc portion of an immunoglobulinmolecule. The Fc region can be, e.g., an Fc region from an IgG1, IgG2,IgG3, IgG4, IgA, IgM, IgE, or IgD immunoglobulin molecule or acombination of portions of each of these. To be clear, the anti-C5aantibodies described herein can be, e.g., of IgG1, IgG2, IgG3, IgG4,IgA, IgM, IgE, or IgD isotype. The amino acid sequences for a number ofhuman heavy chain constant regions are known in the art and describedin, e.g., Kabat et al. (1991), supra.

In some embodiments, the heavy chain polypeptide can comprise a hybridconstant region, or a portion thereof, such as a G2/G4 hybrid constantregion (see e.g., Burton et al. (1992) Adv Immun 51:1-18; Canfield etal. (1991) J Exp Med 173:1483-1491; and Mueller et al. (1997) MolImmunol 34(6):441-452). For example (and in accordance with Kabatnumbering), the IgG1 and IgG4 constant regions comprise G₂₄₉G₂₅₀residues whereas the IgG2 constant region does not comprise residue 249,but does comprise G₂₅₀. In a G2/G4 hybrid constant region, where the249-250 region comes from the G2 sequence, the constant region can befurther modified to introduce a glycine residue at position 249 toproduce a G2/G4 fusion having G₂₄₉/G₂₅₀. Other constant domain hybridsthat comprise G₂₄₉/G₂₅₀ can also be part of engineered antibodies inaccordance with the disclosure. Exemplary heavy chain polypeptide aminoacid sequences are set forth in Table 2.

The anti-C5a antibody can be, e.g., one of the specific antibodiesexemplified in the working examples: BNJ364, BNJ367, BNJ378, BNJ366,BNJ369, BNJ383, BNJ371, or BNJ381. The amino acid sequences for theseexemplified anti-C5a antibodies, which can be used in conjunction withany of the methods described herein, are set forth in Table 2.

TABLE 2 Amino Acid Sequences for Select Humanized Anti-C5a AntibodiesSIN: Ab Description Amino Acid Sequence 16 BNJ364 Full lightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 BNJ364 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSchainGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 18 BNJ364 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 19 BNJ364Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSvariable GSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ364 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ364 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ364 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ364 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 24BNJ364 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAINPNSGGTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWsequence withGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSsignal peptideSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 25BNJ364 Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFK chainDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSSASTKGPSVFPLsequenceAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTwithout signalYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEpeptideDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 26 BNJ364 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 27 BNJ364Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFKvariable DRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSS regionsequence 28 BNJ364 Heavy chain DYSMD variable region sequence KabatHCDR1 29 BNJ364 Heavy chain AINPNSGGTNYNQKFKD variable region sequenceKabat HCDR2 30 BNJ364 Heavy chain SGSYDGYYAMDY variable region sequenceKabat HCDR3 31 BNJ364 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSsequenceNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16 BNJ367 FulllightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 BNJ367 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSchainGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 18 BNJ367 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 19 BNJ367Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSvariable GSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ367 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ367 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ367 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ367 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 32BNJ367 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAINPNSGGTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWsequence withGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSsignal peptideSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 33BNJ367 Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFK chainDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSSASTKGPSVFPLsequenceAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTwithout signalYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEpeptideDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ367 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 27 BNJ367Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFKvariable DRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSS regionsequence 28 BNJ367 Heavy chain DYSMD variable region sequence KabatHCDR1 29 BNJ367 Heavy chain AINPNSGGTNYNQKFKD variable region sequenceKabat HCDR2 30 BNJ367 Heavy chain SGSYDGYYAMDY variable region sequenceKabat HCDR3 34 BNJ367 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 35 BNJ371 FulllightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 36 BNJ371 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYWASTRESGVPDRFSGchainSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCsequenceLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLwithout signal SSPVTKSFNRGEC peptide 18 BNJ371 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 37 BNJ371Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYWASTRESGVPDRFSGvariable SGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ371 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR138 BNJ371 Light chain WASTRES variable region sequence Kabat LCDR2 22BNJ371 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ371 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 32BNJ371 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAINPNSGGTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWsequence withGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSsignal peptideSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 33BNJ371 Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFK chainDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSSASTKGPSVFPLsequenceAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTwithout signalYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEpeptideDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ371 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 27 BNJ371Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFKvariable DRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSS regionsequence 28 BNJ371 Heavy chain DYSMD variable region sequence KabatHCDR1 29 BNJ371 Heavy chain AINPNSGGTNYNQKFKD variable region sequenceKabat HCDR2 30 BNJ371 Heavy chain SGSYDGYYAMDY variable region sequenceKabat HCDR3 34 BNJ371 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 39 BNJ378 FulllightMDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGchainKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKRTVAsequence withAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTsignal peptide LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 40 BNJ378 Full lightDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSchainGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 41 BNJ378 Light chainMDMRVPAQLLGLLLLWLRGARC variable region sequence signal peptide 42 BNJ378Light chainDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSvariable GSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ378 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ378 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ378 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ378 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 32BNJ378 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAINPNSGGTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWsequence withGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSsignal peptideSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 33BNJ378 Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFK chainDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSSASTKGPSVFPLsequenceAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTwithout signalYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEpeptideDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ378 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 27 BNJ378Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAINPNSGGTNYNQKFKvariable DRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGSYDGYYAMDYWGQGTTVTVSS regionsequence 28 BNJ378 Heavy chain DYSMD variable region sequence KabatHCDR1 29 BNJ378 Heavy chain AINPNSGGTNYNQKFKD variable region sequenceKabat HCDR2 30 BNJ378 Heavy chain SGSYDGYYAMDY variable region sequenceKabat HCDR3 34 BNJ378 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 16 BNJ366 FulllightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 BNJ366 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSchainGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 18 BNJ366 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 19 BNJ366Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSvariable GSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ366 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ366 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ366 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ366 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 43BNJ366 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAIHLNTGYTNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGsequence withQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsignal peptideGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44 BNJ366Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFK chainGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSSASTKGPSVFPLAsequencePCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYwithout signalTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPpeptideEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 26 BNJ366 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 45 BNJ366Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFKvariable GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSS regionsequence 28 BNJ366 Heavy chain DYSMD variable region sequence KabatHCDR1 46 BNJ366 Heavy chain AIHLNTGYTNYNQKFKG variable region sequenceKabat HCDR2 47 BNJ366 Heavy chain GFYDGYSPMDY variable region sequenceKabat HCDR3 31 BNJ366 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSsequenceNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16 BNJ369 FulllightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 BNJ369 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSchainGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 18 BNJ369 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 19 BNJ369Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSvariable GSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ369 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ369 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ369 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ369 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 48BNJ369 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAIHLNTGYTNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGsequence withQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsignal peptideGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 49 BNJ369Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFK chainGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSSASTKGPSVFPLAsequencePCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYwithout signalTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPpeptideEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ369 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 45 BNJ369Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFKvariable GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSS regionsequence 28 BNJ369 Heavy chain DYSMD variable region sequence KabatHCDR1 46 BNJ369 Heavy chain AIHLNTGYTNYNQKFKG variable region sequenceKabat HCDR2 47 BNJ369 Heavy chain GFYDGYSPMDY variable region sequenceKabat HCDR3 34 BNJ369 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 35 BNJ381 FulllightMVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPchainKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPsequence withSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSsignal peptide KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 36 BNJ381 Full lightDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKWYWASTRESGVPDRFSG chainSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCsequenceLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLwithout signal SSPVTKSFNRGEC peptide 18 BNJ381 Light chainMVLQTQVFISLLLWISGAYG variable region sequence signal peptide 37 BNJ381Light chainDIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQPPKLLIYWASTRESGVPDRFSGvariable SGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ381 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR138 BNJ381 Light chain WASTRES variable region sequence Kabat LCDR2 22BNJ381 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ381 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 48BNJ381 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAIHLNTGYTNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGsequence withQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsignal peptideGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 49 BNJ381Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFK chainGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSSASTKGPSVFPLAsequencePCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYwithout signalTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPpeptideEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ381 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 45 BNJ381Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFKvariable GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSS regionsequence 28 BNJ381 Heavy chain DYSMD variable region sequence KabatHCDR1 46 BNJ381 Heavy chain AIHLNTGYTNYNQKFKG variable region sequenceKabat HCDR2 47 BNJ381 Heavy chain GFYDGYSPMDY variable region sequenceKabat HCDR3 34 BNJ381 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 39 BNJ383 FulllightMDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGchainKAPKLLIYRASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKRTVAsequence withAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTsignal peptide LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 40 BNJ383 Full lightDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSchainGSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLsequenceLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSwithout signal SPVTKSFNRGEC peptide 41 BNJ383 Light chainMDMRVPAQLLGLLLLWLRGARC variable region sequence signal peptide 42 BNJ383Light chainDIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSvariable GSGTDFTLTISSLQPEDFATYYCQQSNEDPYTFGGGTKVEIKR region sequence 20BNJ383 Light chain RASESVDSYGNSFMH variable region sequence Kabat LCDR121 BNJ383 Light chain RASNLES variable region sequence Kabat LCDR2 22BNJ383 Light chain QQSNEDPYT variable region sequence Kabat LCDR3 23BNJ383 Light chainTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSconstant STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC region sequence 48BNJ383 Full heavyMDWTWRVFCLLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLE chainWMGAIHLNTGYTNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGsequence withQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsignal peptideGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 49 BNJ383Full heavyQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFK chainGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSSASTKGPSVFPLAsequencePCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYwithout signalTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPpeptideEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26 BNJ383 Heavy chainMDWTWRVFCLLAVAPGAHS variable region sequence signal peptide 45 BNJ383Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYSMDWVRQAPGQGLEWMGAIHLNTGYTNYNQKFKvariable GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGFYDGYSPMDYWGQGTTVTVSS regionsequence 28 BNJ383 Heavy chain DYSMD variable region sequence KabatHCDR1 46 BNJ383 Heavy chain AIHLNTGYTNYNQKFKG variable region sequenceKabat HCDR2 47 BNJ383 Heavy chain GFYDGYSPMDY variable region sequenceKabat HCDR3 34 BNJ383 Heavy chainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstantTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVregion TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSsequenceNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK “SIN” refers toSEQ ID NO. “Ab” in Table 2 refers to the alphanumeric designationassigned to a given antibody. Each of the antibodies is exemplified inthe working examples.

In some embodiments, an anti-C5a antibody described herein comprises aChothia-defined light chain CDR set or a combined Kabat-Chothia-definedlight chain CDR set obtained from any of the light chain variableregions described in Tables 2 or 3. In some embodiment, an anti-C5aantibody, or antigen-binding fragment thereof, described hereincomprises a Chothia-defined heavy chain CDR set or a combinedKabat-Chothia-defined heavy chain CDR set obtained from any of the heavychain variable regions described in Tables 2 or 3.

In preferred embodiments, an anti-C5a antibody described herein binds toC5a, but not to native, full-length C5. In some embodiments, an anti-C5aantibody binds to C5a, but does not bind to the alpha chain ofuncleaved, native C5. As used herein, “uncleaved C5” refers to a C5protein that has not been cleaved into fragments C5a and C5b by an AP orCP C5 convertase. An exemplary amino acid sequence for a human C5 alphachain is set forth in Haviland et al. (1991), supra, the sequence ofwhich is incorporated herein by reference in its entirety.

In some embodiments, an anti-C5a antibody described herein does not bindto paralogs of human C5 such as human C3a or human C4a.

The disclosure also features antibodies that crossblock binding of ananti-C5a antibody described herein (e.g., crossblocks any one of BNJ364,BNJ367, BNJ378, BNJ366, BNJ369, BNJ371, BNJ381, or BNJ383). As usedherein, the term “crossblocking antibody” refers to an antibody thatlowers the amount of binding (or prevents binding) of an anti-C5aantibody to an epitope on a complement component C5a protein relative tothe amount of binding of the anti-C5a antibody to the epitope in theabsence of the crossblocking antibody. Suitable methods for determiningwhether a first antibody crossblocks binding of a second antibody to anepitope are known in the art. For example, crossblocking antibodies canbe identified by comparing the binding of the BNJ364 monoclonal anti-C5aantibody in the presence and absence of a test antibody. Decreasedbinding of the BNJ364 antibody in the presence of the test antibody ascompared to binding of the BNJ364 antibody in the absence of the testantibody indicates the test antibody is a crossblocking antibody.

In some embodiments, the binding of an antibody to C5a can inhibit thebiological activity of C5a. Methods for measuring C5a activity include,e.g., chemotaxis assays, radioimmunoassays (RIAs), or enzyme-linkedimmunospecific assays (ELISA) (see, e.g., Ward and Zvaifler (1971) JClin Invest 50(3):606-16 and Wurzner et al. (1991) Complement Inflamm8:328-340). In some embodiments, the binding of an antibody orantigen-binding fragment thereof to C5a can inhibit C5a-mediatedneutrophil activation in vitro. Suitable methods for determining whetheran anti-C5a antibody inhibits C5a-mediated neutrophil activation invitro, or the extent to which the antibody inhibits activation, areknown in the art and exemplified in the working examples below. Forexample, human neutrophils obtained from healthy donors can be isolatedand contacted with isolated human C5a in the presence or absence of atest anti-C5a antibody. C5a-dependent activation of human neutrophilscan be measured as a function of myeloperoxidase (MPO) release from thecells in the presence of C5a. An inhibition of the amount of MPOreleased from the cells in the presence of C5a and the test antibody, ascompared to the amount of MPO released from cells in the presence of C5aand a control antibody, indicates that the test antibody inhibitsC5a-mediated neutrophil activation.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof does not inhibit (or does not substantially inhibit) theactivity of complement component C5, as compared to the level ofinhibition (if any) observed by a corresponding control antibody orantigen-binding fragment thereof (i.e., an antibody that does not bindto free C5a or C5). C5 activity can be measured as a function of itscell-lysing ability in a subject's body fluids. The cell-lysing ability,or a reduction thereof, of C5 can be measured by methods well known inthe art such as, for example, by a conventional hemolytic assay such asthe hemolysis assay described by Kabat and Mayer (eds), “ExperimentalImmunochemistry, 2^(nd) Edition,” 135-240, Springfield, Ill., C C Thomas(1961), pages 135-139, or a conventional variation of that assay such asthe chicken erythrocyte hemolysis method as described in, e.g., Hillmenet al. (2004) N Engl J Med 350(6):552.

In some embodiments, C5 activity, or inhibition thereof, is quantifiedusing a CH50eq assay. The CH50eq assay is a method for measuring thetotal classical complement activity in serum. This test is a lyticassay, which uses antibody-sensitized erythrocytes as the activator ofthe classical complement pathway and various dilutions of the test serumto determine the amount required to give 50% lysis (CH50). The percenthemolysis can be determined, for example, using a spectrophotometer. TheCH50eq assay provides an indirect measure of terminal complement complex(TCC) formation, since the TCC themselves are directly responsible forthe hemolysis that is measured.

The assay is well known and commonly practiced by those of skill in theart. Briefly, to activate the classical complement pathway, undilutedserum samples (e.g., human serum samples) are added to microassay wellscontaining the antibody-sensitized erythrocytes to thereby generate TCC.Next, the activated sera are diluted in microassay wells, which arecoated with a capture reagent (e.g., an antibody that binds to one ormore components of the TCC). The TCC present in the activated samplesbind to the monoclonal antibodies coating the surface of the microassaywells. The wells are washed and, to each well, is added a detectionreagent that is detectably labeled and recognizes the bound TCC. Thedetectable label can be, e.g., a fluorescent label or an enzymaticlabel. The assay results are expressed in CH50 unit equivalents permilliliter (CH50 U Eq/mL).

Additional methods for detecting and/or measuring C5 activity in vitroare set forth and exemplified in the working examples.

In some embodiments, the binding of an antibody to C5a can inhibit theinteraction between C5a and C5aR1. Suitable methods for detecting and/ormeasuring the interaction between C5a and C5aR1 (in the presence andabsence of an antibody) are known in the art and described in, e.g.,Mary and Boulay (1993) Eur J Haematol 51(5):282-287; Kaneko et al.(1995) Immunology 86(1):149-154; Giannini et al. (1995) J Biol Chem270(32):19166-19172; and U.S. Patent Application Publication No.20060160726. For example, the binding of detectably labeled (e.g.,radioactively labeled) C5a to C5aR1-expressing peripheral bloodmononuclear cells can be evaluated in the presence and absence of anantibody. A decrease in the amount of detectably-labeled C5a that bindsto C5aR1 in the presence of the antibody, as compared to the amount ofbinding in the absence of the antibody, is an indication that theantibody inhibits the interaction between C5a and C5aR1.

In some embodiments, the binding of an antibody to C5a can inhibit theinteraction between C5a and C5L2. Methods for detecting and/or measuringthe interaction between C5a and C5L2 are known in the art and describedin, e.g., Ward (2009) J Mol Med 87(4):375-378 and Chen et al. (2007)Nature 446(7132):203-207. Additional methods for evaluating thebiological effect of an anti-C5a antibody described herein areexemplified in the working examples below.

In some embodiments, the anti-C5a antibody specifically binds to a humancomplement component C5a protein (e.g., the human C5a protein having theamino acid sequence depicted in SEQ ID NO:1 or SEQ ID NO:2). The terms“specific binding,” “specifically binds,” and like grammatical terms, asused herein, refer to two molecules forming a complex (e.g., a complexbetween an antibody and a complement component C5a protein) that isrelatively stable under physiologic conditions. Typically, binding isconsidered specific when the association constant (k_(a)) is higher than10⁶ M⁻¹s⁻¹. Thus, an antibody can specifically bind to a C5a proteinwith a k_(a) of at least (or greater than) 10⁶ (e.g., at least orgreater than 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵higher) M⁻¹s⁻¹. In some embodiments, an anti-C5a antibody describedherein has a dissociation constant (k_(d)) of less than or equal to 10⁻³(e.g., 8×10⁻⁴, 5×10⁻⁴, 2×10⁻⁴, 10⁻⁴, or 10⁻⁵) s⁻¹.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M. The equilibriumconstant K_(D) is the ratio of the kinetic rate constants—k_(d)/k_(a).In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 1.25×10⁻⁹ M. Examples of anti-C5a antibodies that bind toC5a with a K_(D) that is less than 1.25×10⁻⁹ M include, e.g., theBNJ364, BNJ367, BNJ371, BNJ378, BNJ366, BNJ369, BNJ381, and BNJ383anti-C5a antibodies.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 1×10⁻⁹ M. Examples of anti-C5a antibodies that bind to C5awith a K_(D) that is less than 10⁻⁹ M include, e.g., the BNJ364, BNJ367,BNJ378, BNJ366, BNJ369, BNJ381, and BNJ383 anti-C5a antibodies.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 5×10⁻¹⁰ M. Examples of anti-C5a antibodies that bind to C5awith a K_(D) that is less than 5×10⁻¹⁰ M include, e.g., the BNJ367,BNJ378, BNJ366, BNJ369, BNJ381, and BNJ383 anti-C5a antibodies.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 2×10⁻¹⁰ M. Examples of anti-C5a antibodies that bind to C5awith a K_(D) that is less than 2×10⁻¹⁰ M include, e.g., the BNJ367,BNJ366, BNJ369, BNJ381, and BNJ383 anti-C5a antibodies.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 1×10⁻¹⁰ M. Examples of anti-C5a antibodies that bind to C5awith a K_(D) that is less than 1×10⁻¹⁰ M include, e.g., the BNJ369,BNJ381, and BNJ383 anti-C5a antibodies.

In some embodiments, an anti-C5a antibody described herein has a K_(D)of less than 7.5×10⁻¹¹ M. Examples of anti-C5a antibodies that bind toC5a with a K_(D) that is less than 7.5×10⁻¹¹ M include, e.g., the BNJ369and BNJ383 anti-C5a antibodies.

Methods for determining the affinity of an antibody for a proteinantigen are known in the art. For example, the affinity of an antibodyfor a protein antigen can be quantified using a variety of techniquessuch as, but not limited to, Western blot, dot blot, BiolayerInterferometry, Surface Plasmon Resonance (SPR) method (e.g., BIAcoresystem; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.),or enzyme-linked immunospecific assays (ELISA). See, e.g., Harlow andLane (1988) “Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo (2004)“Antibody Engineering: Methods and Protocols,” Humana Press (ISBN:1588290921); Borrebaek (1992) “Antibody Engineering, A Practical Guide,”W.H. Freeman and Co., NY; Borrebaek (1995) “Antibody Engineering,”2^(nd) Edition, Oxford University Press, NY, Oxford; Johne et al. (1993)J Immunol Meth 160:191-198; Jonsson et al. (1993) Ann Biol Clin51:19-26; and Jonsson et al. (1991) Biotechniques 11:620-627.

Any of the light chain CDR sets or light chain variable regionsdescribed herein can be paired with any of the heavy chain CDR sets orheavy chain variable regions described herein. It is well within thepurview of the ordinarily skilled artisan to, e.g., confirm (test) thatan anti-C5a antibody generated by such a pairing possesses the desiredaffinity or activity. Suitable methods for confirming the activityand/or affinity of an anti-C5a antibody are described herein.

In some embodiments, the anti-C5a antibodies described herein bind toboth human C5a (hC5a) and C5a from a non-human mammal such as anon-human primate (e.g., cynomolgus macaque). In some embodiments, ananti-C5a antibody or antigen-binding fragment thereof described hereindoes not bind to paralogs of human C5a such as C3a or C4a from the samenon-human mammalian species.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof described herein binds to free hC5a and a cynomolgus macaque C5aprotein comprising, or consisting of, the following amino acid sequence:MLQEKIEEIAAKYKHLVVKK CCYDGVRINH DETCEQRAAR ISVGPRCVKAFTECCVVASQLRANNSHKDLQLGR (SEQ ID NO:179). In some embodiments, ananti-C5a antibody or antigen-binding fragment thereof described hereinbinds to free hC5a and a rhesus macaque C5a protein comprising, orconsisting of, the amino acid sequence depicted in SEQ ID NO:179.

In some embodiments, an antibody, or an antigen-binding fragmentthereof, can bind to a desarginated form of C5a protein from a non-humanmammalian species (e.g., a non-human primate species). For example, theantibody or antigen-binding fragment thereof can bind to a freeC5a-desarg protein from cynomolgus macaque or rhesus macaque, theprotein comprising, or consisting of, the following amino acid sequence:MLQEKIEEIAAKYKHLVVKK CCYDGVRINH DETCEQRAAR ISVGPRCVKAFTECCVVASQLRANNSHKDLQLG (SEQ ID NO:180).

In some embodiments, the anti-C5a antibodies described herein bind tomouse C5a (i.e., the free C5a from mouse). In some embodiments, theanti-C5a antibodies described herein bind to mouse C5a, but not to humanC5a. In some embodiments, an anti-C5a antibody described herein does notbind to uncleaved, native (fully-folded) mouse C5. In some embodiments,an anti-C5a antibody described herein does not bind to paralogs of mouseC5a such as mouse C3a or mouse C4a.

An anti-mouse C5a antibody, or an antigen-binding fragment thereof, canbind to a mouse C5a protein comprising, or consisting of, the followingamino acid sequence:LRQKIEEQAAKYKHSVPKKCCYDGARVNFYETCEERVARVTIGPLCIRAFNECCTIANKIRKESPHKPVQLGR (SEQ ID NO:51). See also, e.g., Wetsel et al. (1987)Biochem 26:737-743. In some embodiments, an anti-mouse C5a antibody, oran antigen-binding fragment thereof, can bind to a desarginated form ofmouse C5a protein comprising, or consisting of, the following amino acidsequence: LRQKIEEQAAKYKHSVPKKCCYDGARVNFYETCEERVARVTIGPLCIRAFNECCTIANKIRKESPHKPVQLG (SEQ ID NO:52). In some embodiments, the anti-mouseC5a antibody binds to both the full-length mouse C5a protein and thedesarginated form of the mouse C5a protein.

An anti-mouse C5a antibody described herein can, e.g., contain a lightchain CDR set obtained from a light chain variable region polypeptidecomprising the following amino acid sequence:EIVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGVGTKLELKR (SEQ ID NO:53). Forexample, an anti-mouse C5a antibody can contain: (i) a Kabat-definedlight chain CDR1 comprising, or consisting of, the following amino acidsequence: RASSSVNYIY (SEQ ID NO:54); (ii) a Kabat-defined light chainCDR2 comprising, or consisting of, the following amino acid sequence:YTSNLAP (SEQ ID NO:55); and/or (iii) a Kabat-defined light chain CDR3comprising, or consisting of, the following amino acid sequence:QQFTSSPLT (SEQ ID NO:56).

The anti-mouse C5a antibody can contain a light chain constant region,e.g., the mouse kappa light chain constant region comprising, orconsisting of, the following amino acid sequence:ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:57).

In some embodiments, an anti-mouse C5a antibody described herein cancontain an amino-terminal signal peptide, e.g., a signal peptidecomprising, or consisting of, the following amino acid sequence:MGWSCIILFLVATATGVHS (SEQ ID NO:58).

In some embodiments, an anti-mouse C5a antibody described herein cancontain a light chain polypeptide comprising, or consisting of, thefollowing amino acid sequence:REIVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGVGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:59) orMGWSCIILFLVATATGVHSREIVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPLTFGVGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC(SEQ ID NO:60). In some embodiments, an anti-mouse C5a antibodydescribed herein contains a light chain polypeptide comprising aminoacids 2 to 214 of SEQ ID NO:59. In some embodiments, an anti-mouse C5aantibody described herein contains a light chain polypeptide comprisingamino acids 1 to 19 and 21 to 233 of SEQ ID NO:60.

An anti-mouse C5a antibody described herein can, e.g., contain a heavychain CDR set obtained from a heavy chain variable region polypeptidecomprising the following amino acid sequence:LEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYINWVKQSHGKSLEWIGYIYPNDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARPYYSDYGM DYWGQGTSVTVSS (SEQID NO:61). For example, an anti-mouse C5a antibody can contain: (i) aKabat-defined heavy chain CDR1 comprising, or consisting of, thefollowing amino acid sequence: DYYYIN (SEQ ID NO:62); (ii) aKabat-defined heavy chain CDR2 comprising, or consisting of, thefollowing amino acid sequence: YIYPNDGDTNYNQKFKG (SEQ ID NO:63); and/or(iii) a Kabat-defined heavy chain CDR3 comprising, or consisting of, thefollowing amino acid sequence: PYYSDYGMDY (SEQ ID NO:64).

The anti-mouse C5a antibody can contain a heavy chain constant region.In some embodiments, an anti-mouse C5a antibody described herein cancontain an amino-terminal signal peptide, e.g., a signal peptidecomprising, or consisting of, the following amino acid sequence:MGWSCIILFLVATATGVHS (SEQ ID NO:65).

In some embodiments, an anti-mouse C5a antibody described herein cancontain a heavy chain polypeptide comprising, or consisting of, thefollowing amino acid sequence:LEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYINWVKQSHGKSLEWIGYIYPNDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARPYYSDYGM DYWGQGTSVTVSS (SEQID NO:66) or MGWSCIILFLVATATGVHSLEVQLQQSGPELVKPGASVKISCKASGYTFTDYYYINWVKQSHGKSLEWIGYIYPNDGDTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARPYYSDYGMDYWGQGTSVTVSS (SEQ ID NO:67). In some embodiments, ananti-mouse C5a antibody described herein contains a heavy chainpolypeptide comprising amino acids 2 to 121 of SEQ ID NO:66. In someembodiments, an anti-mouse C5a antibody described herein contains aheavy chain polypeptide comprising amino acids 1 to 19 and 21 to 140 ofSEQ ID NO:67. In some embodiments, an anti-mouse C5a antibody describedherein contains a heavy chain constant region polypeptide comprising oneor more amino acid substitutions from the above described sequence.

In some embodiments, an anti-mouse C5a antibody described hereincontains a light chain polypeptide comprising: (i) a light chain CDR1comprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:54; (ii) a light chain CDR2 comprising, or consisting of, the aminoacid sequence depicted in SEQ ID NO:55; and (iii) a light chain CDR3comprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:56; (iv) a heavy chain CDR1 comprising, or consisting of, the aminoacid sequence depicted in SEQ ID NO:62; (v) a heavy chain CDR2comprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:63; and/or (vi) a heavy chain CDR3 comprising, or consisting of, theamino acid sequence depicted in SEQ ID NO:64.

In some embodiments, an anti-mouse C5a antibody described hereincontains a light chain polypeptide comprising, or consisting of, theamino acid sequence depicted in SEQ ID NO:59 and a heavy chainpolypeptide comprising, or consisting of, the amino acid sequencedepicted in SEQ ID NO:66.

In some embodiments, an anti-C5a antibody described herein can bind tohuman C5a and to mouse C5a.

Methods for Producing the Anti-C5a Antibodies and Antigen-BindingFragments Thereof

The disclosure also features methods for producing any of the anti-C5aantibodies or antigen-binding fragments thereof described herein. Insome embodiments, methods for preparing an antibody described herein caninclude immunizing a subject (e.g., a non-human mammal) with anappropriate immunogen. Suitable immunogens for generating any of theantibodies described herein are set forth herein. For example, togenerate an antibody that binds to C5a, a skilled artisan can immunize asuitable subject (e.g., a non-human mammal such as a rat, a mouse, agerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-humanprimate) with a full-length C5a polypeptide such as a full-length C5apolypeptide comprising the amino acid sequence depicted in SEQ ID NO:1or the desarginated form of C5a (e.g., the human C5a desarg comprisingthe amino acid sequence depicted in SEQ ID NO:2). In some embodiments,the non-human mammal is C5 deficient, e.g., a C5-deficient mousedescribed in, e.g., Levy and Ladda (1971) Nat New Biol 229(2):51-52;Crocker et al. (1974) J Clin Pathol 27(2):122-124; Wetsel et al. (1990)J Biol Chem 265:2435-2440; and Jungi and Pepys (1981) Immunology43(2):271-279. Human C5a can be purified from human serum as describedin, e.g., McCarthy and Henson (1979) J Immunol 123(6):2511-2517 andManderino et al. (1982) J Immunol Methods 53(1):41-50. See also theworking examples. Human C5a can also be generated in vitro as describedin, e.g., Vallota and Müller-Eberhard (1973) J Exp Med 137:1109.Purified human C5a is also commercially available from, e.g., ComplementTechnology, Inc. (catalog number A144; Tyler, Tex.). Recombinant C5a canalso be generated by one of ordinary skill in the art as described in,e.g., Tothe et al. (1994) Prot Sci 3:1159-1168.

A suitable subject (e.g., a non-human mammal) can be immunized with theappropriate antigen along with subsequent booster immunizations a numberof times sufficient to elicit the production of an antibody by themammal. The immunogen can be administered to a subject (e.g., anon-human mammal) with an adjuvant. Adjuvants useful in producing anantibody in a subject include, but are not limited to, proteinadjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvum or Salmonella minnesota) and bacterial componentsincluding cell wall skeleton, trehalose dimycolate, monophosphoryl lipidA, methanol extractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g.,aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Otheradjuvants that can be used in the methods for inducing an immuneresponse include, e.g., cholera toxin and parapoxvirus proteins. Seealso Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g.,Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J MedChem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Guptaet al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, the methods include preparing a hybridoma cell linethat secretes a monoclonal antibody that binds to the immunogen. Forexample, a suitable mammal such as a laboratory mouse is immunized witha C5a polypeptide as described above. Antibody-producing cells (e.g., Bcells of the spleen) of the immunized mammal can be isolated two to fourdays after at least one booster immunization of the immunogen and thengrown briefly in culture before fusion with cells of a suitable myelomacell line. The cells can be fused in the presence of a fusion promotersuch as, e.g., vaccinia virus or polyethylene glycol. The hybrid cellsobtained in the fusion are cloned, and cell clones secreting the desiredantibodies are selected. For example, spleen cells of Balb/c miceimmunized with a suitable immunogen can be fused with cells of themyeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. After thefusion, the cells are expanded in suitable culture medium, which issupplemented with a selection medium, for example HAT medium, at regularintervals in order to prevent normal myeloma cells from overgrowing thedesired hybridoma cells. The obtained hybrid cells are then screened forsecretion of the desired antibodies, e.g., an antibody that binds to C5aand inhibits the interaction between C5a and a C5a receptor (e.g.,C5aR1).

In some embodiments, a skilled artisan can identify an anti-C5a antibodyfrom a non-immune biased library as described in, e.g., U.S. Pat. No.6,300,064 (to Knappik et al.; Morphosys A G) and Schoonbroodt et al.(2005) Nucleic Acids Res 33(9):e81.

In some embodiments, the methods described herein can involve, or beused in conjunction with, e.g., phage display technologies, bacterialdisplay, yeast surface display, eukaryotic viral display, mammalian celldisplay, and cell-free (e.g., ribosomal display) antibody screeningtechniques (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935;Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000)Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder etal. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) CombChem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel etal. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) NatBiotechnol 18:1287-1292).

Methods for identifying antibodies using various phage display methodsare known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. Such phage can be utilized todisplay antigen-binding domains of antibodies, such as Fab, Fv, ordisulfide-bond stabilized Fv antibody fragments, expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage used in these methods are typically filamentous phage such as fdand M13. The antigen binding domains are expressed as a recombinantlyfused protein to any of the phage coat proteins pIII, pVIII, or pIX.See, e.g., Shi et al. (2010) JMB 397:385-396. Examples of phage displaymethods that can be used to make the immunoglobulins, or fragmentsthereof, described herein include those disclosed in Brinkman et al.(1995) J Immunol Methods 182:41-50; Ames et al. (1995) J Immunol Methods184:177-186; Kettleborough et al. (1994) Eur J Immunol 24:952-958;Persic et al. (1997) Gene 187:9-18; Burton et al. (1994) Advances inImmunology 57:191-280; and PCT publication nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO95/20401. Suitable methods are also described in, e.g., U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108.

In some embodiments, the phage display antibody libraries can begenerated using mRNA collected from B cells from the immunized mammals.For example, a splenic cell sample comprising B cells can be isolatedfrom mice immunized with C5a polypeptide as described above. mRNA can beisolated from the cells and converted to cDNA using standard molecularbiology techniques. See, e.g., Sambrook et al. (1989) “MolecularCloning: A Laboratory Manual, 2^(nd) Edition,” Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988),supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995), supra. ThecDNA coding for the variable regions of the heavy chain and light chainpolypeptides of immunoglobulins are used to construct the phage displaylibrary. Methods for generating such a library are described in, e.g.,Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro et al.(2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) MethodsMol Biol 51:355-376.

In some embodiments, a combination of selection and screening can beemployed to identify an antibody of interest from, e.g., a population ofhybridoma-derived antibodies or a phage display antibody library.Suitable methods are known in the art and are described in, e.g.,Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al.(1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994),supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. Forexample, a plurality of phagemid vectors, each encoding a fusion proteinof a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage)and a different antigen-combining region are produced using standardmolecular biology techniques and then introduced into a population ofbacteria (e.g., E. coli). Expression of the bacteriophage in bacteriacan, in some embodiments, require use of a helper phage. In someembodiments, no helper phage is required (see, e.g., Chasteen et al.(2006) Nucleic Acids Res 34(21):e145). Phage produced from the bacteriaare recovered and then contacted to, e.g., a target antigen bound to asolid support (immobilized). Phage may also be contacted to antigen insolution, and the complex is subsequently bound to a solid support.

In some embodiments, the immobilized phage are the phage of interest.Accordingly, the unbound phage are removed by washing the support.Following the wash step, bound phage are then eluted from the solidsupport, e.g., using a low pH buffer or a free target antigencompetitor, and recovered by infecting bacteria. In some embodiments,the phage that are not immobilized are the phage of interest. In suchembodiments, the population of phage can be contacted to the antigen twoor more times to deplete from the population any of the phage that bindto the support. Unbound phage are then collected and used for subsequentscreening steps.

To enrich the phage population for phage particles that containantibodies having a higher affinity for the target antigen (whilereducing the proportion of phage that may bind to the antigennon-specifically), the eluted phage (described above) can be used tore-infect a population of bacterial host cells. The expressed phage arethen isolated from the bacteria and again contacted to a target antigen.The concentration of antigen, pH, temperature and inclusion ofdetergents and adjuvants during contact can be modulated to enrich forhigher affinity antibody fragments. The unbound phage are removed bywashing the solid support. The number or cycles, duration, pH,temperature and inclusion of detergents and adjuvants during washing canalso be modulated to enrich for higher affinity antibody fragments.Following the wash step, bound phage are then eluted from the solidsupport. Anywhere from one to six iterative cycles of panning may beused to enrich for phage containing antibodies having higher affinityfor the target antigen. In some embodiments, a deselection step can alsobe performed in conjunction with any of the panning approaches describedherein.

Individual phage of the population can be isolated by infecting bacteriaand then plating at a density to allow formation of monoclonalantibodies.

For example, to identify using phage display techniques an antibody thatbinds to C5a, but not to C5, the following panning approach can beemployed. The population can first be contacted to a surface containingbound native, full-length human C5. The process can be repeated two ormore times, each time collecting the unbound phage. The population canalso be contacted to a solid support containing surface-bound C4 and/orC3 proteins. Unbound phage from the foregoing steps are then contactedto a surface containing bound C5a or desarginated C5a. Phage that bindto C5a are eluted from the surface and recovered by infecting bacteria.Iterative rounds of phage selection may be performed. After one to sixrounds of selection, individual recovered phagemid can be screened forexpression of antibody fragments with the desired specificity andaffinity.

A subpopulation of antibodies screened using the above methods can becharacterized for their specificity and binding affinity for aparticular immunogen (e.g., C5a) using any immunological or biochemicalbased method known in the art. For example, specific binding of anantibody to C5a, as compared to native, full-length C5, may bedetermined for example using immunological or biochemical based methodssuch as, but not limited to, an ELISA assay, SPR assays,immunoprecipitation assay, affinity chromatography, and equilibriumdialysis as described above. Immunoassays which can be used to analyzeimmunospecific binding and cross-reactivity of the antibodies include,but are not limited to, competitive and non-competitive assay systemsusing techniques such as Western blots, RIA, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays. Such assays are routine andwell known in the art.

Antibodies can also be assayed using any SPR-based assays known in theart for characterizing the kinetic parameters of the interaction of theantibody with C5a. Any SPR instrument commercially available including,but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden);lAsys instruments (Affinity Sensors; Franklin, Mass.); IBIS system(Windsor Scientific Limited; Berks, UK), SPR-CELLIA systems (NipponLaser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta(Texas Instruments; Dallas, Tex.) can be used in the methods describedherein. See, e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al.(2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) CurrOpin Biotechnol 9: 97-101; and Rich et al (2000) Curr Opin Biotechnol11: 54-61.

It is understood that the above methods can also be used to determineif, e.g., an anti-C5a antibody does not bind to full-length, native C5,C3, and/or C4 proteins. The above methods can also be used to determineif an antibody that binds to C5a also inhibits the interaction betweenC5a and a C5a receptor. The above methods can also be used to determineif an antibody that binds to C5a also inhibits the activity of C5a.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any desiredfragments, and expressed in any desired host, including mammalian cells,insect cells, plant cells, yeast, and bacteria, e.g., as described indetail below. For example, techniques to recombinantly produce Fab, Fab′and F(ab′)₂ fragments can also be employed using methods known in theart such as those disclosed in PCT publication no. WO 92/22324; Mullinaxet al. (1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) Am JRepr Immunol 34:26-34; and Better et al. (1988) Science 240:1041-1043.Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu etal. (1993) Proc Nat Acad Sci USA 90:7995-7999; and Skerra et al. (1988)Science 240:1038-1040.

Phage display technology can also be used to, e.g., increase theaffinity of an antibody for its cognate antigen. The technology,referred to as affinity maturation, can employ mutagenesis or CDRwalking and re-selection to identify antibodies that bind with higheraffinity to an antigen as compared to the initial or parental antibody.See, e.g., Glaser et al. (1992) J Immunol 149:3903-3913. Libraries canbe constructed consisting of a pool of variant clones, each differing byone or more amino acid substitutions. Mutants with increased bindingaffinity for the antigen can be selected for by contacting theimmobilized mutants with labeled antigen or any combination of methodsdescribed above. Any screening method known in the art can be used toidentify mutant antibodies with increased affinity to the antigen (e.g.,SPR or ELISA techniques).

In some embodiments, epitope mapping can be used to identify, e.g., theregion of C5a that interacts with an antibody, e.g., a region of C5athat binds to C5aR1. Methods for identifying the epitope to which aparticular antibody binds are also known in the art and are describedabove.

The antibodies and fragments thereof identified herein can be or can bemade “chimeric.” Chimeric antibodies and antigen-binding fragmentsthereof comprise portions from two or more different species (e.g.,mouse and human). Chimeric antibodies can be produced with mousevariable regions of desired specificity fused to human constant domains(for example, U.S. Pat. No. 4,816,567). In this manner, non-humanantibodies can be modified to make them more suitable for human clinicalapplication (e.g., methods for treating or preventing acomplement-mediated disorder in a subject).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (see, e.g., Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536), by substituting rodent frameworksor CDR sequences for the corresponding sequences of a human antibody.Also see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In someembodiments, humanized forms of non-human (e.g., mouse) antibodies arehuman antibodies (recipient antibody) in which the CDR region amino acidresidues of the non-human antibody (e.g., mouse, rat, rabbit, ornon-human primate antibody) having the desired specificity, affinity,and binding capacity are grafted onto the framework scaffold of a humanantibody. Additional humanization methods are described below in theworking examples.

Methods for grafting CDR sequences from a donor antibody (e.g., anon-human antibody) to the framework regions of an acceptor antibody(e.g., a human antibody) are well known in the art and are described in,e.g., Jones et al. (1986) Nature 321:522-525; Verhoeyen et al. (1988)Science 239(4847):1534-1536; Riechmann et al. (1988) Nature 332:323-327;Queen et al. (1989) Proc Natl Acad Sci USA 86:10029-10033; PCTpublication no. WO 93/011237; Kettleborough et al. (1991) ProteinEngineering, Design and Selection 4:773-783; Benny K. C. Lo (2004)“Antibody Engineering: Methods and Protocols,” Humana Press (ISBN:1588290921); Borrebaek (1992) “Antibody Engineering, A Practical Guide,”W.H. Freeman and Co., NY; and Borrebaek (1995) “Antibody Engineering,”2^(nd) Edition, Oxford University Press, NY, Oxford. For example, CDRsfrom a donor antibody can be grafted onto framework regions of anacceptor antibody using overlap extension polymerase chain reaction(PCR) techniques as described in, e.g., Daugherty et al. (1991) NucleicAcids Res 19(9):2471-2476; Roguska et al. (1996) Protein Engineering9(10):895-904; and Yazaki et al. (2004) Protein Engineering, Design &Selection 17(5):481-489.

In embodiments where the selected CDR amino acid sequences are shortsequences (e.g., fewer than 10-15 amino acids in length), nucleic acidsencoding the CDRs can be chemically synthesized as described in, e.g.,Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 andU.S. Pat. No. 6,995,259. For a given nucleic acid sequence encoding anacceptor antibody, the region of the nucleic acid sequence encoding theCDRs can be replaced with the chemically synthesized nucleic acids usingstandard molecular biology techniques. The 5′ and 3′ ends of thechemically synthesized nucleic acids can be synthesized to comprisesticky end restriction enzyme sites for use in cloning the nucleic acidsinto the nucleic acid encoding the variable region of the donorantibody.

In some instances, one or more framework region amino acid residues ofthe human immunoglobulin are also replaced by corresponding amino acidresidues of the non-human antibody (so called “back mutations”). Inaddition, phage display libraries can be used to vary amino acids atchosen positions within the antibody sequence. The properties of ahumanized antibody are also affected by the choice of the humanframework. Furthermore, humanized and chimerized antibodies can bemodified to comprise residues that are not found in the recipientantibody or in the donor antibody in order to further improve antibodyproperties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human immunoglobulin sequences,preferably human germline sequences. Human antibodies can include aminoacid residues not encoded by human germline immunoglobulin sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo). However, the term “humanantibody” does not include antibodies in which CDR sequences derivedfrom another mammalian species, such as a mouse, have been grafted ontohuman framework sequences (i.e., humanized antibodies). Fully human orhuman antibodies may be derived from transgenic mice carrying humanantibody genes (carrying the variable (V), diversity (D), joining (J),and constant (C) exons) or from human cells. For example, it is nowpossible to produce transgenic animals (e.g., mice) that are capable,upon immunization, of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. See, e.g.,Jakobovits et al. (1993) Proc Natl Acad Sci USA 90:2551; Jakobovits etal. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol.7:33; and Duchosal et al. (1992) Nature 355:258. Transgenic mousestrains can be engineered to contain gene sequences from unrearrangedhuman immunoglobulin genes. One example of such a mouse is the HuMAbMouse® (Medarex, Inc.), which contains human immunoglobulin transgeneminiloci that encode unrearranged human μ heavy and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci. See, e.g., Lonberg, et al.(1994) Nature 368(6474):856-859. The preparation and use of HuMab mice,and the genomic modifications carried by such mice, are furtherdescribed in Taylor et al. (1992) Nucleic Acids Res 20:6287-6295; Chen,J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al.(1993) Proc Natl Acad Sci USA 90:3720-3724; Choi et al. (1993) NatureGenetics 4:1 17-123; Tuaillon et al. (1994) J Immunol 152:2912-2920;Taylor et al. (1994) International Immunology 6:579-591; and Fishwild etal. (1996) Nature Biotechnol 14:845-851. An alternative transgenic mousesystem for expressing human immunoglobulin genes is referred to as theXenomouse (Abgenix, Inc.) and is described in, e.g., U.S. Pat. Nos.6,075,181; 6,114,598; 6,150,584; and 6,162,963. Like the HuMAb Mouse®system, the Xenomouse system involves disruption of the endogenous mouseheavy and light chain genes and insertion into the genome of the mousetransgenes carrying unrearranged human heavy and light chainimmunoglobulin loci that contain human variable and constant regionsequences. Other systems known in the art for expressing humanimmunoglobulin genes include the KM Mouse® system, described in detailin PCT Publication WO 02/43478 and the TC mouse system described inTomizuka et al. (2000) Proc Natl Acad Sci USA 97:722-727.

The human sequences may code for both the heavy and light chains ofhuman antibodies and would function correctly in the mice, undergoingrearrangement to provide a wide antibody repertoire similar to that inhumans. The transgenic mice can be immunized with the target proteinimmunogen to create a diverse array of specific antibodies and theirencoding RNA. Nucleic acids encoding the antibody chain components ofsuch antibodies may then be cloned from the animal into a displayvector. Typically, separate populations of nucleic acids encoding heavyand light chain sequences are cloned, and the separate populations thenrecombined on insertion into the vector, such that any given copy of thevector receives a random combination of a heavy and a light chain. Thevector is designed to express antibody chains so that they can beassembled and displayed on the outer surface of a display packagecontaining the vector. For example, antibody chains can be expressed asfusion proteins with a phage coat protein from the outer surface of thephage. Thereafter, display packages can be selected and screened fordisplay of antibodies binding to a target.

In addition, the phage-display libraries screened above can includehuman antibodies (Hoogenboom et al. (1992) J Mol Biol 227:381; Marks etal. (1991) J Mol Biol 222:581-597; and Vaughan et al. (1996) NatureBiotech 14:309). Synthetic phage libraries can be created which userandomized combinations of synthetic human antibody V-regions. Byselection on antigen, fully human antibodies can be made in which theV-regions are very human-like in nature. See, e.g., U.S. Pat. Nos.6,794,132; 6,680,209; 4,634,666; and Ostberg et al. (1983) Hybridoma2:361-367, the contents of each of which are incorporated herein byreference in their entirety.

For the generation of human antibodies, also see Mendez et al. (1998)Nature Genetics 15:146-156 and Green and Jakobovits (1998) J Exp Med188:483-495, the disclosures of which are hereby incorporated byreference in their entirety. Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181;6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. PatentPublication Nos. 20030229905 A1, 20040010810 A1, 20040093622 A1,20060040363 A1, 20050054055 A1, 20050076395 A1, and 20050287630 A1. Seealso International Publication Nos. WO 94/02602, WO 96/34096, and WO98/24893, and European Patent No. EP 0 463 151 B1. The disclosures ofeach of the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825;5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, thedisclosures of which are hereby incorporated by reference in theirentirety. See also European Patent No. 0 546 073 B1, InternationalPatent Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO97/13852, and WO 98/24884, the disclosures of each of which are herebyincorporated by reference in their entirety. See further Taylor et al.(1992) Nucleic Acids Res 20: 6287; Chen et al. (1993) Int Immunol 5:647; Tuaillon et al. (1993) Proc Natl Acad Sci USA 90: 3720-4; Choi etal. (1993) Nature Genetics 4: 117; Lonberg et al. (1994) Nature 368:856-859; Taylor et al. (1994) International Immunology 6: 579-591;Tuaillon et al. (1995) J. Immunol 154: 6453-65; Fishwild et al. (1996)Nature Biotechnology 14: 845; and Tuaillon et al. (2000) Eur J Immunol.10: 2998-3005, the disclosures of each of which are hereby incorporatedby reference in their entirety.

In certain embodiments, de-immunized forms of the antibodies, orantigen-binding fragments described herein are provided. De-immunizedantibodies or antigen-binding fragments thereof are antibodies that havebeen modified so as to render the antibody or antigen-binding fragmentthereof non-immunogenic, or less immunogenic, to a given species.De-immunization can be achieved by modifying the antibody orantigen-binding fragment thereof utilizing any of a variety oftechniques known to those skilled in the art (see, e.g., PCT PublicationNos. WO 04/108158 and WO 00/34317). For example, an antibody orantigen-binding fragment thereof may be de-immunized by identifyingpotential T cell epitopes and/or B cell epitopes within the amino acidsequence of the antibody or antigen-binding fragment thereof andremoving one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized antibodiesdescribed herein include de-immunized antigen-binding fragments, Fab,Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies, murine antibodies,fully human antibodies, engineered antibodies (such as, for example,chimeric, single chain, CDR-grafted, humanized, and artificiallyselected antibodies), synthetic antibodies and semi-syntheticantibodies.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for C5a, the other one is for anyother antigen.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavychain/light-chain pairs have different specificities (Milstein andCuello (1983) Nature 305:537-539). Antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) can befused to immunoglobulin constant domain sequences. The fusion of theheavy chain variable region is preferably is with an immunoglobulinheavy-chain constant domain, including at least part of the hinge,C_(H)2, and C_(H)3 regions. DNAs encoding the immunoglobulin heavy-chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. For further details of illustrative currently knownmethods for generating bispecific antibodies see, e.g., Suresh et al.(1986) Methods in Enzymology 121:210; PCT Publication No. WO 96/27011;Brennan et al. (1985) Science 229:81; Shalaby et al., J Exp Med (1992)175:217-225; Kostelny et al. (1992) J Immunol 148(5):1547-1553;Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber etal. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60.Bispecific antibodies also include cross-linked or heteroconjugateantibodies. Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al. (1992) J Immunol148(5):1547-1553. The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See, e.g., Gruber et al. (1994) J Immunol 152:5368.Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies)are contemplated and described in, e.g., Tutt et al. (1991) J Immunol147:60.

The disclosure also embraces variant forms of multi-specific antibodiessuch as the dual variable domain immunoglobulin (DVD-Ig) moleculesdescribed in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297. TheDVD-Ig molecules are designed such that two different light chainvariable domains (VL) from two different parent antibodies are linked intandem directly or via a short linker by recombinant DNA techniques,followed by the light chain constant domain. Similarly, the heavy chaincomprises two different heavy chain variable domains (VH) linked intandem, followed by the constant domain C_(H)1 and Fc region. Methodsfor making DVD-Ig molecules from two parent antibodies are furtherdescribed in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715.

The disclosure also provides camelid or dromedary antibodies (e.g.,antibodies derived from Camelus bactrianus, Calelus dromaderius, or lamapaccos). Such antibodies, unlike the typical two-chain (fragment) orfour-chain (whole antibody) antibodies from most mammals, generally lacklight chains. See U.S. Pat. No. 5,759,808; Stijlemans et al. (2004) JBiol Chem 279:1256-1261; Dumoulin et al. (2003) Nature 424:783-788; andPleschberger et al. (2003) Bioconjugate Chem 14:440-448. Engineeredlibraries of camelid antibodies and antibody fragments are commerciallyavailable, for example, from Ablynx (Ghent, Belgium). As with otherantibodies of non-human origin, an amino acid sequence of a camelidantibody can be altered recombinantly to obtain a sequence that moreclosely resembles a human sequence, i.e., the nanobody can be“humanized” to thereby further reduce the potential immunogenicity ofthe antibody.

In some embodiments, the anti-C5a antibodies described herein comprisean altered heavy chain constant region that has reduced (or no) effectorfunction relative to its corresponding unaltered constant region.Effector functions involving the constant region of the anti-C5aantibody may be modulated by altering properties of the constant or Fcregion. Altered effector functions include, for example, a modulation inone or more of the following activities: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis,binding to one or more Fc-receptors, and pro-inflammatory responses.Modulation refers to an increase, decrease, or elimination of aneffector function activity exhibited by a subject antibody containing analtered constant region as compared to the activity of the unalteredform of the constant region. In particular embodiments, modulationincludes situations in which an activity is abolished or completelyabsent.

An altered constant region with altered FcR binding affinity and/or ADCCactivity and/or altered CDC activity is a polypeptide which has eitheran enhanced or diminished FcR binding activity and/or ADCC activityand/or CDC activity compared to the unaltered form of the constantregion. An altered constant region which displays increased binding toan FcR binds at least one FcR with greater affinity than the unalteredpolypeptide. An altered constant region which displays decreased bindingto an FcR binds at least one FcR with lower affinity than the unalteredform of the constant region. Such variants which display decreasedbinding to an FcR may possess little or no appreciable binding to anFcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1%) of the binding to the FcR as compared to the level ofbinding of a native sequence immunoglobulin constant or Fc region to theFcR. Similarly, an altered constant region that displays modulated ADCCand/or CDC activity may exhibit either increased or reduced ADCC and/orCDC activity compared to the unaltered constant region. For example, insome embodiments, the anti-C5a antibody comprising an altered constantregion can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activityof the unaltered form of the constant region. An anti-C5a antibodydescribed herein comprising an altered constant region displayingreduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDCactivity as exemplified herein.

In certain embodiments, the altered constant region has at least oneamino acid substitution, insertion, and/or deletion, compared to anative sequence constant region or to the unaltered constant region,e.g. from about one to about one hundred amino acid substitutions,insertions, and/or deletions in a native sequence constant region or inthe constant region of the parent polypeptide. In some embodiments, thealtered constant region herein will possess at least about 70% homology(similarity) or identity with the unaltered constant region and in someinstances at least about 75% and in other instances at least about 80%homology or identity therewith, and in other embodiments at least about85%, 90% or 95% homology or identity therewith. The altered constantregion may also contain one or more amino acid deletions or insertions.Additionally, the altered constant region may contain one or more aminoacid substitutions, deletions, or insertions that results in alteredpost-translational modifications, including, for example, an alteredglycosylation pattern (e.g., the addition of one or more sugarcomponents, the loss of one or more sugar components, or a change incomposition of one or more sugar components relative to the unalteredconstant region).

Antibodies with altered or no effector functions may be generated byengineering or producing antibodies with variant constant, Fc, or heavychain regions; recombinant DNA technology and/or cell culture andexpression conditions may be used to produce antibodies with alteredfunction and/or activity. For example, recombinant DNA technology may beused to engineer one or more amino acid substitutions, deletions, orinsertions in regions (such as, for example, Fc or constant regions)that affect antibody function including effector functions.Alternatively, changes in post-translational modifications, such as,e.g., glycosylation patterns, may be achieved by manipulating the cellculture and expression conditions by which the antibody is produced.Suitable methods for introducing one or more substitutions, additions,or deletions into an Fc region of an antibody are well known in the artand include, e.g., standard DNA mutagenesis techniques as described in,e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,2^(nd) Edition,” Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane (1988), supra; Borrebaek (1992), supra;Johne et al. (1993), supra; PCT publication no. WO 06/53301; and U.S.Pat. No. 7,704,497.

In some embodiments, an anti-C5a antibody described herein exhibitsreduced or no effector function. In some embodiments, an anti-C5aantibody comprises a hybrid constant region, or a portion thereof, suchas a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) AdvImmun 51:1-18; Canfield et al. (1991) J Exp Med 173:1483-1491; andMueller et al. (1997) Mol Immunol 34(6):441-452). See above.

In addition to using a G2/G4 construct as described above, an anti-C5aantibody described herein having reduced effector function may beproduced by introducing other types of changes in the amino acidsequence of certain regions of the antibody. Such amino acid sequencechanges include but are not limited to the Ala-Ala mutation describedin, e.g., PCT Publication nos. WO 94/28027 and WO 98/47531; and Xu etal. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, ananti-C5a antibody with one or more mutations within the constant regionincluding the Ala-Ala mutation has reduced or no effector function.According to these embodiments, the constant region of the antibody cancomprise a substitution to an alanine at position 234 or a mutation toan alanine at position 235. Additionally, the altered constant regionmay contain a double mutation: a mutation to an alanine at position 234and a second mutation to an alanine at position 235. In one embodiment,an anti-C5a antibody comprises an IgG4 framework, wherein the Ala-Alamutation would describe a mutation(s) from phenylalanine to alanine atposition 234 and/or a mutation from leucine to alanine at position 235.In another embodiment, the anti-C5a antibody comprises an IgG1framework, wherein the Ala-Ala mutation would describe a mutation(s)from leucine to alanine at position 234 and/or a mutation from leucineto alanine at position 235. An anti-C5a antibody may alternatively oradditionally carry other mutations, including the point mutation K322Ain the CH2 domain (Hezareh et al. (2001) J Virol 75:12161-12168). Anantibody with said mutation(s) in the constant region may furthermore bea blocking or non-blocking antibody.

Additional substitutions that, when introduced into a heavy chainconstant region, result in decreased effector function are set forth in,e.g., Shields et al. (2001) J Biol Chem 276(9):6591-6604. Seeparticularly Table 1 (“Binding of human IgG1 variants to human FcRn andFcγR) of Shields et al., the disclosure of which is incorporated hereinby reference in its entirety. By screening a library of anti-IgEantibodies, each antibody of the library differing by one or moresubstitutions in the heavy chain constant region, for binding to a panelof Fc receptors (including FcRn, FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA),the authors identified a number of substitutions that modulate specificFc-Fc receptor interactions. For example, a variant IgG2a heavy chainconstant region in which the CH2 domain contains a D265A substitution(heavy chain amino acid numbering according to Kabat et al. (supra))results in a complete loss of interaction between the variant constantregion and IgG Fc receptors FcγRIIB, FcγRIII, FcγRI, and FcγRIV. Shieldset al. (2001) at page 6595, Table 1. See also Baudino et al. (2008) JImmunol 181:6664-6669 (supra).

Changes within the hinge region also affect effector functions. Forexample, deletion of the hinge region may reduce affinity for Fcreceptors and may reduce complement activation (Klein et al. (1981) ProcNatl Acad Sci USA 78: 524-528). The present disclosure therefore alsorelates to antibodies with alterations in the hinge region.

In some embodiments, an anti-C5a antibody may contain an alteredconstant region exhibiting enhanced or reduced complement dependentcytotoxicity (CDC). Modulated CDC activity may be achieved byintroducing one or more amino acid substitutions, insertions, ordeletions in an Fc region of the antibody. See, e.g., U.S. Pat. No.6,194,551. Alternatively or additionally, cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved or reduced internalization capability and/or increased ordecreased complement-mediated cell killing. See, e.g., Caron et al.(1992) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922;PCT publication nos. WO 99/51642 and WO 94/29351; Duncan and Winter(1988) Nature 322:738-40; and U.S. Pat. Nos. 5,648,260 and 5,624,821.

Another potential means of modulating effector function of antibodiesincludes changes in glycosylation, which is summarized in, e.g., Raju(2003) BioProcess International 1(4):44-53. According to Wright andMorrison, the microheterogeneity of human IgG oligosaccharides canaffect biological functions such as CDC and ADCC, binding to various Fcreceptors, and binding to Clq protein. (1997) TIBTECH 15:26-32.Glycosylation patterns of antibodies can differ depending on theproducing cell and the cell culture conditions (Raju, supra). Suchdifferences can lead to changes in both effector function andpharmacokinetics. See, e.g., Israel et al. (1996) Immunology89(4):573-578; Newkirk et al. (1996) Clin Exp Immunol 106(2):259-264.Differences in effector function may be related to the IgG's ability tobind to the Fcγ receptors (FcγRs) on the effector cells. Shields et al.have shown that IgG, with alterations in amino acid sequence that haveimproved binding to FcγR, can exhibit up to 100% enhanced ADCC usinghuman effector cells. (2001) J Biol Chem 276(9):6591-6604. While thesealterations include changes in amino acids not found at the bindinginterface, both the nature of the sugar component as well as itsstructural pattern may also contribute to the differences observed. Inaddition, the presence or absence of fucose in the oligosaccharidecomponent of an IgG can improve binding and ADCC. See, e.g., Shields etal. (2002) J Biol Chem 277(30):26733-26740. An IgG that lacked afucosylated carbohydrate linked to Asn²⁹⁷ exhibited normal receptorbinding to the FcγRI receptor. In contrast, binding to the FcγRIIIAreceptor was improved 50-fold and accompanied by enhanced ADCC,especially at lower antibody concentrations.

Shinkawa et al. demonstrated that an antibody to the human IL-5 receptorproduced in a rat hybridoma showed more than 50% higher ADCC whencompared to the antibody produced in Chinese hamster ovary cells (CHO)(Shinkawa et al. (2003) J Biol Chem 278(5):3466-73). Monosaccharidecomposition and oligosaccharide profiling showed that the rathybridoma-produced IgG had a lower content of fucose than theCHO-produced protein. The authors concluded that the lack offucosylation of an IgG1 has a critical role in enhancement of ADCCactivity.

A different approach was taken by Umana et al. who changed theglycosylation pattern of chCE7, a chimeric IgG1 anti-neuroblastomaantibody. (1999) Nat Biotechnol 17(2):176-180). Using tetracycline, theyregulated the activity of a glycosyltransferase enzyme (GnTIII) whichbisects oligosaccharides that have been implicated in ADCC activity. TheADCC activity of the parent antibody was barely above background level.Measurement of ADCC activity of the chCE7 produced at differenttetracycline levels showed an optimal range of GnTIII expression formaximal chCE7 in vitro ADCC activity. This activity correlated with thelevel of constant region-associated, bisected complex oligosaccharide.Newly optimized variants exhibited substantial ADCC activity. Similarly,Wright and Morrison produced antibodies in a CHO cell line deficient inglycosylation and showed that antibodies produced in this cell line wereincapable of complement-mediated cytolysis. (1994) J Exp Med180:1087-1096. Thus, as known alterations that affect effector functioninclude modifications in the glycosylation pattern or a change in thenumber of glycosylated residues, the present disclosure relates to ananti-C5a antibody wherein glycosylation is altered to either enhance ordecrease effector function(s) including ADCC and CDC. Alteredglycosylation includes a decrease or increase in the number ofglycosylated residues as well as a change in the pattern or location ofglycosylated residues.

Still other approaches exist for altering the effector function ofantibodies. For example, antibody-producing cells can be hypermutagenic,thereby generating antibodies with randomly altered polypeptide residuesthroughout an entire antibody molecule. See, e.g., PCT publication no.WO 05/011735. Hypermutagenic host cells include cells deficient in DNAmismatch repair. Antibodies produced in this manner may be lessantigenic and/or have beneficial pharmacokinetic properties.Additionally, such antibodies may be selected for properties such asenhanced or decreased effector function(s). Additional details ofmolecular biology techniques useful for preparing an antibody orantigen-binding fragment thereof described herein are set forth below.

Recombinant Antibody Expression and Purification

The antibodies or antigen-binding fragments thereof described herein canbe produced using a variety of techniques known in the art of molecularbiology and protein chemistry. For example, a nucleic acid encoding oneor both of the heavy and light chain polypeptides of an antibody can beinserted into an expression vector that contains transcriptional andtranslational regulatory sequences, which include, e.g., promotersequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, transcriptionterminator signals, polyadenylation signals, and enhancer or activatorsequences. The regulatory sequences include a promoter andtranscriptional start and stop sequences. In addition, the expressionvector can include more than one replication system such that it can bemaintained in two different organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain polypeptides from nucleic acids inmammalian cells. One class of vectors relies upon the integration of thedesired gene sequences into the host cell genome. Cells which havestably integrated DNA can be selected by simultaneously introducing drugresistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc NatlAcad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol ApplGenet 1:327). The selectable marker gene can be either linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection (Wigler et al. (1979) Cell 16:77). A second class ofvectors utilizes DNA elements which confer autonomously replicatingcapabilities to an extrachromosomal plasmid. These vectors can bederived from animal viruses, such as bovine papillomavirus (Sarver etal. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitablefor subsequent expression of the nucleic acid. The method ofintroduction is largely dictated by the targeted cell type, discussedbelow. Exemplary methods include CaPO₄ precipitation, liposome fusion,cationic liposomes, electroporation, viral infection, dextran-mediatedtransfection, polybrene-mediated transfection, protoplast fusion, anddirect microinjection.

Appropriate host cells for the expression of antibodies orantigen-binding fragments thereof include yeast, bacteria, insect,plant, and mammalian cells. Of particular interest are bacteria such asE. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris,insect cells such as SF9, mammalian cell lines (e.g., human cell lines),as well as primary cell lines.

In some embodiments, an antibody or fragment thereof can be expressedin, and purified from, transgenic animals (e.g., transgenic mammals).For example, an antibody can be produced in transgenic non-human mammals(e.g., rodents) and isolated from milk as described in, e.g., Houdebine(2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al.(2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J ImmunolMethods 231(1-2):147-157.

The antibodies and fragments thereof can be produced from the cells byculturing a host cell transformed with the expression vector containingnucleic acid encoding the antibodies or fragments, under conditions, andfor an amount of time, sufficient to allow expression of the proteins.Such conditions for protein expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. For example,antibodies expressed in E. coli can be refolded from inclusion bodies(see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expressionsystems and methods for their use are well known in the art (see CurrentProtocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—ALaboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York(2001)). The choice of codons, suitable expression vectors and suitablehost cells will vary depending on a number of factors, and may be easilyoptimized as needed. An antibody (or fragment thereof) described hereincan be expressed in mammalian cells or in other expression systemsincluding but not limited to yeast, baculovirus, and in vitro expressionsystems (see, e.g., Kaszubska et al. (2000) Protein Expression andPurification 18:213-220).

Following expression, the antibodies and fragments thereof can beisolated. The term “purified” or “isolated” as applied to any of theproteins (antibodies or fragments) described herein refers to apolypeptide that has been separated or purified from components (e.g.,proteins or other naturally-occurring biological or organic molecules)which naturally accompany it, e.g., other proteins, lipids, and nucleicacid in a prokaryote expressing the proteins. Typically, a polypeptideis purified when it constitutes at least 60 (e.g., at least 65, 70, 75,80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in asample.

An antibody or fragment thereof can be isolated or purified in a varietyof ways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography. For example, an antibody can bepurified using a standard anti-antibody column (e.g., a protein-A orprotein-G column). Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. See, e.g.,Scopes (1994) “Protein Purification, 3^(rd) edition,” Springer-Verlag,New York City, N.Y. The degree of purification necessary will varydepending on the desired use. In some instances, no purification of theexpressed antibody or fragments thereof will be necessary.

Methods for determining the yield or purity of a purified antibody orfragment thereof are known in the art and include, e.g., Bradford assay,UV spectroscopy, Biuret protein assay, Lowry protein assay, amido blackprotein assay, high pressure liquid chromatography (HPLC), massspectrometry (MS), and gel electrophoretic methods (e.g., using aprotein stain such as Coomassie Blue or colloidal silver stain).

In some embodiments, endotoxin can be removed from the antibodies orfragments. Methods for removing endotoxin from a protein sample areknown in the art and exemplified in the working examples. For example,endotoxin can be removed from a protein sample using a variety ofcommercially available reagents including, without limitation, theProteoSpin™ Endotoxin Removal Kits (Norgen Biotek Corporation),Detoxi-Gel Endotoxin Removal Gel (Thermo Scientific; Pierce ProteinResearch Products), MiraCLEAN® Endotoxin Removal Kit (Minis), orAcrodisc™-Mustang® E membrane (Pall Corporation).

Methods for detecting and/or measuring the amount of endotoxin presentin a sample (both before and after purification) are known in the artand commercial kits are available. For example, the concentration ofendotoxin in a protein sample can be determined using the QCL-1000Chromogenic kit (BioWhittaker), the limulus amebocyte lysate (LAL)-basedkits such as the Pyrotell®, Pyrotell®-T, Pyrochrome®, Chromo-LAL, andCSE kits available from the Associates of Cape Cod Incorporated.

While in no way intended to be limiting, exemplary methods forgenerating the antibodies described herein are set forth in the workingExamples.

Modification of the Antibodies or Antigen-Binding Fragments Thereof

The antibodies or antigen-binding fragments thereof can be modifiedfollowing their expression and purification. The modifications can becovalent or non-covalent modifications. Such modifications can beintroduced into the antibodies or fragments by, e.g., reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Suitable sites for modification can be chosen using any of avariety of criteria including, e.g., structural analysis or amino acidsequence analysis of the antibodies or fragments.

In some embodiments, the antibodies or antigen-binding fragments thereofcan be conjugated to a heterologous moiety. The heterologous moiety canbe, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxinor a drug), or a detectable label such as, but not limited to, aradioactive label, an enzymatic label, a fluorescent label, a heavymetal label, a luminescent label, or an affinity tag such as biotin orstreptavidin. Suitable heterologous polypeptides include, e.g., anantigenic tag (e.g., FLAG (DYKDDDDK (SEQ ID NO:50)), polyhistidine(6-His; HHHHHH (SEQ ID NO:81), hemagglutinin (HA; YPYDVPDYA (SEQ IDNO:82)), glutathione-S-transferase (GST), or maltose-binding protein(MBP)) for use in purifying the antibodies or fragments. Heterologouspolypeptides also include polypeptides (e.g., enzymes) that are usefulas diagnostic or detectable markers, for example, luciferase, afluorescent protein (e.g., green fluorescent protein (GFP)), orchloramphenicol acetyl transferase (CAT). Suitable radioactive labelsinclude, e.g., ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S and ³H. Suitablefluorescent labels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488,phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700,Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any ofa variety of luminescent lanthanide (e.g., europium or terbium)chelates. For example, suitable europium chelates include the europiumchelate of diethylene triamine pentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labelsinclude, e.g., alkaline phosphatase, CAT, luciferase, and horseradishperoxidase.

Two proteins (e.g., an antibody and a heterologous moiety) can becross-linked using any of a number of known chemical cross linkers.Examples of such cross linkers are those which link two amino acidresidues via a linkage that includes a “hindered” disulfide bond. Inthese linkages, a disulfide bond within the cross-linking unit isprotected (by hindering groups on either side of the disulfide bond)from reduction by the action, for example, of reduced glutathione or theenzyme disulfide reductase. One suitable reagent,4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene (SMPT),forms such a linkage between two proteins utilizing a terminal lysine onone of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated tothe amino acid backbone of the antibody. Alternatively, the radioactivelabel can be included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds tofree amino groups to form meta-iodophenyl (mIP) derivatives of relevantproteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) orchelate (e.g., to DOTA or DTPA) which is in turn bound to the proteinbackbone. Methods of conjugating the radioactive labels or largermolecules/chelates containing them to the antibodies or antigen-bindingfragments described herein are known in the art. Such methods involveincubating the proteins with the radioactive label under conditions(e.g., pH, salt concentration, and/or temperature) that facilitatebinding of the radioactive label or chelate to the protein (see, e.g.,U.S. Pat. No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an antibody) are known in the art ofprotein chemistry. For example, fluorophores can be conjugated to freeamino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines)of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP)ester moieties attached to the fluorophores. In some embodiments, thefluorophores can be conjugated to a heterobifunctional cross-linkermoiety such as sulfo-SMCC. Suitable conjugation methods involveincubating an antibody protein, or fragment thereof, with thefluorophore under conditions that facilitate binding of the fluorophoreto the protein. See, e.g., Welch and Redvanly (2003) “Handbook ofRadiopharmaceuticals: Radiochemistry and Applications,” John Wiley andSons (ISBN 0471495603).

In some embodiments, the antibodies or fragments can be modified, e.g.,with a moiety that improves the stabilization and/or retention of theantibodies in circulation, e.g., in blood, serum, or other tissues. Forexample, the antibody or fragment can be PEGylated as described in,e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al.(2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al.(2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (FreseniusKabi, Germany; see, e.g., Pavisić et al. (2010) Int J Pharm387(1-2):110-119). The stabilization moiety can improve the stability,or retention of, the antibody (or fragment) by at least 1.5 (e.g., atleast 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the antibodies or antigen-binding fragments thereofdescribed herein can be glycosylated. In some embodiments, an antibodyor antigen-binding fragment thereof described herein can be subjected toenzymatic or chemical treatment, or produced from a cell, such that theantibody or fragment has reduced or absent glycosylation. Methods forproducing antibodies with reduced glycosylation are known in the art anddescribed in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

Pharmaceutical Compositions

Compositions containing an antibody or an antigen-binding fragmentthereof described herein can be formulated as a pharmaceuticalcomposition, e.g., for administration to a subject for the treatment orprevention of a complement-associated disorder. The pharmaceuticalcompositions will generally include a pharmaceutically acceptablecarrier. As used herein, a “pharmaceutically acceptable carrier” refersto, and includes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66: 1-19).

The compositions can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed in, e.g., Gennaro (2000) “Remington: The Science and Practiceof Pharmacy,” 20^(th) Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and DrugDelivery Systems,” 7^(th) Edition, Lippincott Williams & WilkinsPublishers (ISBN: 0683305727); and Kibbe (2000) “Handbook ofPharmaceutical Excipients American Pharmaceutical Association,” 3^(rd)Edition (ISBN: 091733096X). In some embodiments, a composition can beformulated, for example, as a buffered solution at a suitableconcentration and suitable for storage at 2-8° C. (e.g., 4° C.). In someembodiments, a composition can be formulated for storage at atemperature below 0° C. (e.g., −20° C. or −80° C.). In some embodiments,the composition can be formulated for storage for up to 2 years (e.g.,one month, two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, 10 months, 11 months, 1year, 1½ years, or 2 years) at 2-8° C. (e.g., 4° C.). Thus, in someembodiments, the compositions described herein are stable in storage forat least 1 year at 2-8° C. (e.g., 4° C.).

The pharmaceutical compositions can be in a variety of forms. Theseforms include, e.g., liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends, in part, on the intended mode ofadministration and therapeutic application. For example, compositionscontaining an antibody or fragment intended for systemic or localdelivery can be in the form of injectable or infusible solutions.Accordingly, the compositions can be formulated for administration by aparenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, orintramuscular injection). “Parenteral administration,” “administeredparenterally,” and other grammatically equivalent phrases, as usedherein, refer to modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intranasal, intraocular, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural,intracerebral, intracranial, intracarotid and intrasternal injection andinfusion.

The compositions can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an antibody (or a fragment of the antibody)described herein in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating an antibody or fragment described herein into a sterilevehicle that contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods forpreparation include vacuum drying and freeze-drying that yield a powderof an antibody, or an antigen-binding fragment thereof, described hereinplus any additional desired ingredient (see below) from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition areagent that delays absorption, for example, monostearate salts, andgelatin.

The anti-C5a antibodies, or antigen-binding fragments thereof, describedherein can also be formulated in immunoliposome compositions. Liposomescontaining the antibody can be prepared by methods known in the art suchas, e.g., the methods described in Epstein et al. (1985) Proc Natl AcadSci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030; andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in, e.g., U.S. Pat. No. 5,013,556.

In certain embodiments, an antibody or an antigen-binding fragmentthereof can be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are known in the art. See, e.g., J. R. Robinson(1978) “Sustained and Controlled Release Drug Delivery Systems,” MarcelDekker, Inc., New York.

In some embodiments, an antibody or antigen-binding fragment describedherein can be formulated in a composition suitable for intrapulmonaryadministration (e.g., for administration via nebulizer; see below) to amammal such as a human. Methods for preparing such compositions are wellknown in the art and described in, e.g., U.S. patent applicationpublication no. 20080202513; U.S. Pat. Nos. 7,112,341 and 6,019,968; andPCT application publication nos. WO 00/061178 and WO 06/122257, thedisclosures of each of which are incorporated herein by reference intheir entirety. Dry powder inhaler formulations and suitable systems foradministration of the formulations are described in, e.g., U.S. patentapplication publication no. 20070235029, PCT Publication No. WO00/69887; and U.S. Pat. No. 5,997,848.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof described herein can be formulated in a composition suitable fordelivery to the eye. In some embodiments, one or more of the anti-C5aantibodies (or antigen-binding fragments thereof) described herein canbe administered locally, for example, by way of topical application orintravitreal injection. For example, in some embodiments, the anti-C5aantibodies can be formulated for administration by way of an eye drop.

The therapeutic preparation for treating the eye can contain one or moreof the anti-C5a antibodies in a concentration from about 0.01 to about1% by weight, preferably from about 0.05 to about 0.5% in apharmaceutically acceptable solution, suspension or ointment. Thepreparation will preferably be in the form of a sterile aqueous solutioncontaining, e.g., additional ingredients such as, but not limited to,preservatives, buffers, tonicity agents, antioxidants and stabilizers,nonionic wetting or clarifying agents, and viscosity-increasing agents.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include, e.g., boric acid, sodium and potassiumbicarbonate, sodium and potassium borates, sodium and potassiumcarbonate, sodium acetate, and sodium biphosphate, in amounts sufficientto maintain the pH at between about pH 6 and pH 8, and preferably,between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40,dextran 70, dextrose, glycerin, potassium chloride, propylene glycol,and sodium chloride.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, and thiourea. Suitable wetting andclarifying agents include polysorbate 80, polysorbate 20, poloxamer 282and tyloxapol. Suitable viscosity-increasing agents include dextran 40,dextran 70, gelatin, glycerin, hydroxyethylcellulose,hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, andcarboxymethylcellulose. The preparation can be administered topically tothe eye of the subject in need of treatment (e.g., a subject afflictedwith AMD) by conventional methods, e.g., in the form of drops, or bybathing the eye in a therapeutic solution, containing one or moreanti-C5a antibodies.

In addition, a variety of devices have been developed for introducingdrugs into the vitreal cavity of the eye. For example, U.S. patentapplication publication no. 20020026176 describes apharmaceutical-containing plug that can be inserted through the sclerasuch that it projects into the vitreous cavity to deliver thepharmaceutical agent into the vitreous cavity. In another example, U.S.Pat. No. 5,443,505 describes an implantable device for introduction intoa suprachoroidal space or an avascular region for sustained release ofdrug into the interior of the eye. U.S. Pat. Nos. 5,773,019 and6,001,386 each disclose an implantable drug delivery device attachableto the scleral surface of an eye. The device comprises an inner corecontaining an effective amount of a low solubility agent covered by anon-bioerodible polymer that is permeable to the low solubility agent.During operation, the low solubility agent permeates the bioerodiblepolymer cover for sustained release out of the device. Additionalmethods and devices (e.g., a transscleral patch and delivery via contactlenses) for delivery of a therapeutic agent to the eye are described in,e.g., Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Rantaand Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocas andBalachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10); Gulsen andChauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al. (2007)Ophthalmic Res 39:244-254; and PCT publication no. WO 04/073551, thedisclosures of which are incorporated herein by reference in theirentirety.

Nucleic acids encoding an antibody (or an antigen-binding fragmentthereof) can be incorporated into a gene construct to be used as a partof a gene therapy protocol to deliver nucleic acids that can be used toexpress and produce agents within cells (see below). Expressionconstructs of such components may be administered in any therapeuticallyeffective carrier, e.g., any formulation or composition capable ofeffectively delivering the component gene to cells in vivo. Approachesinclude insertion of the subject gene in viral vectors includingrecombinant retroviruses, adenovirus, adeno-associated virus,lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant bacterialor eukaryotic plasmids. Viral vectors can transfect cells directly;plasmid DNA can be delivered with the help of, for example, cationicliposomes (lipofectin) or derivatized (e.g., antibody conjugated),polylysine conjugates, gramicidin S, artificial viral envelopes or othersuch intracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation (see, e.g., WO04/060407) carried out invivo. (See also, “Ex vivo Approaches,” below.) Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are known to thoseskilled in the art (see, e.g., Eglitis et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA85:6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA 85:3014-3018;Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huberet al. (1991) Proc Natl Acad Sci USA 88:8039-8043; Ferry et al. (1991)Proc Natl Acad Sci USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc Natl Acad Sci USA 89:10892-10895; Hwu et al. (1993) JImmunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCTPublication Nos. WO89/07136, WO89/02468, WO89/05345, and WO92/07573).Another viral gene delivery system utilizes adenovirus-derived vectors(see, e.g., Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155). Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7, etc.) are known to those skilled in the art. Yet another viralvector system useful for delivery of the subject gene is theadeno-associated virus (AAV). See, e.g., Flotte et al. (1992) Am JRespir Cell Mol Biol 7:349-356; Samulski et al. (1989) J Virol63:3822-3828; and McLaughlin et al. (1989) J Virol 62:1963-1973.

In some embodiments, an antibody, or antigen-binding fragment thereof,described herein can be formulated with one or more additional activeagents useful for treating or preventing a complement-associateddisorder in a subject. Additional agents for treating acomplement-associated disorder in a subject will vary depending on theparticular disorder being treated, but can include, without limitation,an antihypertensive (e.g., an angiotensin-converting enzyme inhibitor),an anticoagulant, a corticosteroid (e.g., prednisone), or animmunosuppressive agent (e.g., vincristine or cyclosporine A). Examplesof anticoagulants include, e.g., warfarin (Coumadin), heparin,phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g.,argatroban, lepirudin, bivalirudin, or dabigatran). An antibody orfragment thereof described herein can also be formulated with afibrinolytic agent (e.g., ancrod, ε-aminocaproic acid, antiplasmin-a₁,prostacyclin, and defibrotide) for the treatment of acomplement-mediated disorder. In some embodiments, an antibody can beformulated with a lipid-lowering agent such as an inhibitor ofhydroxymethylglutaryl CoA reductase. In some embodiments, an antibodycan be formulated with, or for use with, an anti-CD20 agent such asrituximab (Rituxan™; Biogen Idec, Cambridge, Mass.). In someembodiments, e.g., for the treatment of RA, the antibody orantigen-binding fragment thereof can be formulated with one or both ofinfliximab (Remicade®; Centocor, Inc.) and methotrexate (Rheumatrex®,Trexall®). In some embodiments, an antibody or an antigen-bindingfragment thereof described herein can be formulated with a non-steroidalanti-inflammatory drug (NSAID). Many different NSAIDS are available,some over the counter including ibuprofen (Advil®, Motrin®, Nuprin®) andnaproxen (Alleve®) and many others are available by prescriptionincluding meloxicam (Mobic®), etodolac (Lodine®), nabumetone (Relafen®),sulindac (Clinoril®), tolementin (Tolectin®), choline magnesiumsalicylate (Trilasate®), diclofenac (Cataflam®, Voltaren®, Arthrotec®),Diflusinal (Dolobid®), indomethicin (Indocin®), Ketoprofen (Orudis®,Oruvail®), oxaprozin (Daypro®), and piroxicam (Feldene®). In someembodiments an antibody or a fragment thereof can be formulated for usewith an anti-hypertensive, an anti-seizure agent (e.g., magnesiumsulfate), or an anti-thrombotic agent. Anti-hypertensives include, e.g.,labetalol, hydralazine, nifedipine, calcium channel antagonists,nitroglycerin, or sodium nitroprussiate. See, e.g., Mihu et al. (2007) JGasrointestin Liver Dis 16(4):419-424. Anti-thrombotic agents include,e.g., heparin, antithrombin, prostacyclin, or low dose aspirin.

In some embodiments, an antibody or antigen-binding fragment thereof canbe formulated for administration to a subject along with intravenousgamma globulin therapy (IVIG), plasmapheresis, or plasma exchange. Insome embodiments, an anti-C5a antibody or antigen-binding fragmentthereof can be formulated for use before, during, or after, a kidneytransplant.

When an antibody or antigen-binding fragment thereof is to be used incombination with a second active agent, the agents can be formulatedseparately or together. For example, the respective pharmaceuticalcompositions can be mixed, e.g., just prior to administration, andadministered together or can be administered separately, e.g., at thesame or different times (see below).

As described above, a composition can be formulated such that itincludes a therapeutically effective amount of an anti-C5a antibody orantigen-binding fragment thereof described herein. In some embodiments,a composition can be formulated to include a sub-therapeutic amount ofthe antibody (or fragment) and a sub-therapeutic amount of one or moreadditional active agents such that the components in total aretherapeutically effective for treating or preventing acomplement-associated disorder. Methods for determining atherapeutically effective dose of an agent such as a therapeuticantibody are known in the art and described herein.

Applications

The antibodies, antigen-binding fragments thereof, conjugates, andcompositions of any of the foregoing can be used in a number ofdiagnostic and therapeutic applications. For example, detectably-labeledanti-C5a antibodies (e.g., anti-human C5a antibodies or anti-mouse C5aantibodies) can be used in assays to detect the presence or amount ofC5a present in a biological sample. Determining the amount of C5a in asample, e.g., a patient blood sample, can be useful to evaluate thelevel of complement activation in the sample. Suitable methods for usingthe antibodies in diagnostic assays are known in the art and include,without limitation, ELISA, fluorescence resonance energy transferapplications, Western blot, and dot blot techniques. See, e.g., Sambrooket al., supra and Ausubel et al., supra.

In some embodiments, the antibodies and antigen-binding fragmentsdescribed herein can be used as positive controls in assays designed toidentify additional novel compounds for treating complement-mediateddisorders. For example, an anti-C5a antibody that inhibits C5a activitycan be used as a positive control in an assay to identify additionalcompounds (e.g., small molecules, aptamers, or antibodies) that inhibitC5a or C5a-dependent C5a receptor signaling.

In some embodiments, the cross-reactive anti-C5a antibodies orantigen-binding fragments thereof (e.g., cross-reactive with human C5aand, e.g., cynomolgus macaque C5a) described herein can be used forpre-clinical testing in non-human mammals, e.g., pharmacokinetic orpharmacodynamic studies in non-human primates. Accordingly, a researcherwishing to evaluate the efficacy of an anti-C5a antibody in treating acomplement-associate disorder of interest (e.g., RA or sepsis) can use across-reactive anti-C5a antibody described herein in an appropriatenon-human primate model of the disease. If the researcher, for example,establishes efficacy of the antibody in the non-human primate model,these results may provide sufficient proof-of-concept support forregulatory approval for use of the antibody in treating humans.Alternatively, or in addition, a researcher may administer thecross-reactive antibody to a non-human primate to study, e.g., antibodyclearance and/or pharmacodynamics properties. Based on such studiesusing the cross-reactive antibody, the researcher can better approximatethe dose required to treat human disease.

In some embodiments, the anti-mouse C5a antibodies or antigen-bindingfragments thereof described herein, as well as antibodies thatcrossreact with human and mouse C5a, can be used as a surrogate antibodyin mouse models of human disease. This can be especially useful where ahumanized anti-human C5a antibody does not crossreact with mouse C5aand/or is likely to cause an anti-human antibody response in a mouse towhich the humanized antibody is administered. Accordingly, a researcherwishing to study the effect of an anti-C5a antibody in treating adisease (e.g., ischemia-reperfusion injury) can use an anti-mouse C5aantibody described herein in an appropriate mouse model of the disease.If the researcher can establish efficacy in the mouse model of diseaseusing the anti-mouse C5a antibody, the results may establishproof-of-concept for use of an anti-human C5a antibody in treating thedisease in humans. The working examples disclose an exemplary studyusing an anti-mouse C5a antibody surrogate in a mouse model of RAestablishing proof-of-concept for the use of an anti-human C5a antibodyto treat RA in man.

The anti-C5a antibodies described herein can also be used in methods forpurifying C5a from a sample (e.g., a biological sample). In someembodiments, an anti-C5a antibody can be immobilized on a solid phasesupport using methods well known in the art. A sample containing theantigen to be purified, in this case C5a, is contacted to the antibodyon the solid support under conditions and for a time sufficient to allowthe antigen to bind to the antibody. The solid support is then washedone or more times with a suitable buffer to remove unbound material. Thesolid support can be then contacted with a second buffer that results inthe release of the antigen from the antibody. The released antigen isthen collected and characterized (e.g., for purity and activity) usingwell known methods in the art.

The anti-C5a antibodies and antigen-binding fragments thereof describedherein can also be used in therapeutic methods as elaborated on below.

Methods for Treatment

The above-described compositions are useful in, inter alia, methods fortreating or preventing a variety of complement-associated disorders in asubject. The compositions can be administered to a subject, e.g., ahuman subject, using a variety of methods that depend, in part, on theroute of administration. The route can be, e.g., intravenous injectionor infusion (IV), subcutaneous injection (SC), intraperitoneal (IP)injection, or intramuscular injection (IM).

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures ofeach of which are incorporated herein by reference in their entirety.The composition can be delivered to the subject by way of an implantabledevice based on, e.g., diffusive, erodible, or convective systems, e.g.,osmotic pumps, biodegradable implants, electrodiffusion systems,electroosmosis systems, vapor pressure pumps, electrolytic pumps,effervescent pumps, piezoelectric pumps, erosion-based systems, orelectromechanical systems.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof is therapeutically delivered to a subject by way of localadministration. As used herein, “local administration” or “localdelivery,” refers to delivery that does not rely upon transport of thecomposition or agent to its intended target tissue or site via thevascular system. For example, the composition may be delivered byinjection or implantation of the composition or agent or by injection orimplantation of a device containing the composition or agent. Followinglocal administration in the vicinity of a target tissue or site, thecomposition or agent, or one or more components thereof, may diffuse tothe intended target tissue or site.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof can be locally administered to a joint (e.g., an articulatedjoint). For example, in embodiments where the complement-associateddisorder is arthritis, the complement inhibitor can be administereddirectly to a joint (e.g., into a joint space) or in the vicinity of ajoint. Examples of intraarticular joints to which an anti-C5a antibodyor antigen-binding fragment thereof can be locally administered include,e.g., the hip, knee, elbow, wrist, sternoclavicular, temperomandibular,carpal, tarsal, ankle, and any other joint subject to arthriticconditions. An anti-C5a antibody or antigen-binding fragment thereof canalso be administered to bursa such as, e.g., acromial, bicipitoradial,cubitoradial, deltoid, infrapatellar, ischial, and any other bursa knownin the art of medicine.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof can be locally administered to the eye. As used herein, the term“eye” refers to any and all anatomical tissues and structures associatedwith an eye. The eye has a wall composed of three distinct layers: theouter sclera, the middle choroid layer, and the inner retina. Thechamber behind the lens is filled with a gelatinous fluid referred to asthe vitreous humor. At the back of the eye is the retina, which detectslight. The cornea is an optically transparent tissue, which conveysimages to the back of the eye. The cornea includes one pathway for thepermeation of drugs into the eye. Other anatomical tissue structuresassociated with the eye include the lacrimal drainage system, whichincludes a secretory system, a distributive system and an excretorysystem. The secretory system comprises secretors that are stimulated byblinking and temperature change due to tear evaporation and reflexsecretors that have an efferent parasympathetic nerve supply and secretetears in response to physical or emotional stimulation. The distributivesystem includes the eyelids and the tear meniscus around the lid edgesof an open eye, which spread tears over the ocular surface by blinking,thus reducing dry areas from developing.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof is administered to the posterior chamber of the eye. In someembodiments, an anti-C5a antibody or antigen-binding fragment thereof isadministered intravitreally. In some embodiments, an anti-C5a antibodyor antigen-binding fragment thereof is administered trans-sclerally.

In some embodiments, e.g., in embodiments for treatment or prevention ofa complement-associated pulmonary disorder such as COPD or asthma, ananti-C5a antibody or antigen-binding fragment thereof described hereincan also be administered to a subject by way of the lung. Pulmonary drugdelivery may be achieved by inhalation, and administration by inhalationherein may be oral and/or nasal. Examples of pharmaceutical devices forpulmonary delivery include metered dose inhalers, dry powder inhalers(DPIs), and nebulizers. For example, an anti-C5a antibody or anantigen-binding fragment thereof can be administered to the lungs of asubject by way of a dry powder inhaler. These inhalers arepropellant-free devices that deliver dispersible and stable dry powderformulations to the lungs. Dry powder inhalers are well known in the artof medicine and include, without limitation: the TurboHaler®(AstraZeneca; London, England) the AIR® inhaler (Alkermes®; Cambridge,Mass.); Rotahaler® (GlaxoSmithKline; London, England); and Eclipse™(Sanofi-Aventis; Paris, France). See also, e.g., PCT Publication Nos. WO04/026380, WO 04/024156, and WO 01/78693. DPI devices have been used forpulmonary administration of polypeptides such as insulin and growthhormone. In some embodiments, an anti-C5a antibody or an antigen-bindingfragment thereof can be intrapulmonarily administered by way of ametered dose inhaler. These inhalers rely on a propellant to deliver adiscrete dose of a compound to the lungs. Examples of compoundsadministered by metered dose inhalers include, e.g., Astovent®(Boehringer-Ingelheim; Ridgefield, Conn.) and Flovent®(GlaxoSmithKline). See also, e.g., U.S. Pat. Nos. 6,170,717; 5,447,150;and 6,095,141.

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof can be administered to the lungs of a subject by way of anebulizer. Nebulizers use compressed air to deliver a compound as aliquefied aerosol or mist. A nebulizer can be, e.g., a jet nebulizer(e.g., air or liquid-jet nebulizers) or an ultrasonic nebulizer.Additional devices and intrapulmonary administration methods are setforth in, e.g., U.S. Patent Application Publication Nos. 20050271660 and20090110679, the disclosures of each of which are incorporated herein byreference in their entirety.

In some embodiments, the antibodies or antigen-binding fragments thereofprovided herein are present in unit dosage form, which can beparticularly suitable for self-administration. A formulated product ofthe present disclosure can be included within a container, typically,for example, a vial, cartridge, prefilled syringe or disposable pen. Adoser such as the doser device described in U.S. Pat. No. 6,302,855 mayalso be used, for example, with an injection system of the presentdisclosure.

An injection system of the present disclosure may employ a delivery penas described in U.S. Pat. No. 5,308,341. Pen devices, most commonly usedfor self-delivery of insulin to patients with diabetes, are well knownin the art. Such devices can comprise at least one injection needle(e.g., a 31 gauge needle of about 5 to 8 mm in length), are typicallypre-filled with one or more therapeutic unit doses of a therapeuticsolution, and are useful for rapidly delivering the solution to asubject with as little pain as possible.

One medication delivery pen includes a vial holder into which a vial ofinsulin or other medication may be received. The vial holder is anelongate generally tubular structure with proximal and distal ends. Thedistal end of the vial holder includes mounting means for engaging adouble-ended needle cannula. The proximal end also includes mountingmeans for engaging a pen body which includes a driver and dose settingapparatus. A disposable medication (e.g., a high concentration solutionof an anti-C5a antibody or antigen-binding fragment thereof) containingvial for use with the prior art vial holder includes a distal end havinga pierceable elastomeric septum that can be pierced by one end of adouble-ended needle cannula. The proximal end of this vial includes astopper slidably disposed in fluid tight engagement with the cylindricalwall of the vial. This medication delivery pen is used by inserting thevial of medication into the vial holder. A pen body then is connected tothe proximal end of the vial holder. The pen body includes a dosesetting apparatus for designating a dose of medication to be deliveredby the pen and a driving apparatus for urging the stopper of the vialdistally for a distance corresponding to the selected dose. The user ofthe pen mounts a double-ended needle cannula to the distal end of thevial holder such that the proximal point of the needle cannula piercesthe septum on the vial. The patient then selects a dose and operates thepen to urge the stopper distally to deliver the selected dose. The doseselecting apparatus returns to zero upon injection of the selected dose.The patient then removes and discards the needle cannula, and keeps themedication delivery pen in a convenient location for the next requiredmedication administration. The medication in the vial will becomeexhausted after several such administrations of medication. The patientthen separates the vial holder from the pen body. The empty vial maythen be removed and discarded. A new vial can be inserted into the vialholder, and the vial holder and pen body can be reassembled and used asexplained above. Accordingly, a medication delivery pen generally has adrive mechanism for accurate dosing and ease of use.

A dosage mechanism such as a rotatable knob allows the user toaccurately adjust the amount of medication that will be injected by thepen from a prepackaged vial of medication. To inject the dose ofmedication, the user inserts the needle under the skin and depresses theknob once as far as it will depress. The pen may be an entirelymechanical device or it may be combined with electronic circuitry toaccurately set and/or indicate the dosage of medication that is injectedinto the user. See U.S. Pat. No. 6,192,891.

In some embodiments, the needle of the pen device is disposable and thekits include one or more disposable replacement needles. Pen devicessuitable for delivery of the any one of the presently featuredantibodies or antigen-binding fragments thereof are also described in,e.g., U.S. Pat. Nos. 6,277,099; 6,200,296; and 6,146,361, thedisclosures of each of which are incorporated herein by reference intheir entirety. A microneedle-based pen device is described in, e.g.,U.S. Pat. No. 7,556,615, the disclosure of which is incorporated hereinby reference in its entirety. See also the Precision Pen Injector (PPI)device, Molly™, manufactured by Scandinavian Health Ltd.

The present disclosure also presents controlled-release orextended-release formulations suitable for chronic and/orself-administration of a medication such as an anti-C5a antibody or anantigen-binding fragment thereof described herein. The variousformulations can be administered to a patient in need of treatment withthe medication as a bolus or by continuous infusion over a period oftime.

In some embodiments, a high concentration anti-C5a antibody (orantigen-binding fragment thereof) described herein is formulated forsustained-release, extended-release, timed-release, controlled-release,or continuous-release administration. In some embodiments, depotformulations are used to administer the antibody to the subject in needthereof. In this method, the antibody is formulated with one or morecarriers providing a gradual release of active agent over a period of anumber of hours or days. Such formulations are often based upon adegrading matrix which gradually disperses in the body to release theactive agent.

In some embodiments, a C5a-binding fragment (e.g., a single chainantibody, a diabody, or a Fab′ fragment) of an anti-C5a antibodydescribed herein is administered by way of intrapulmonary administrationto a subject in need thereof. For example, a single chain antibody formof any of the anti-C5a antibodies described herein can be delivered byway of a nebulizer or an inhaler to a subject (e.g., a human) afflictedwith a complement-associated pulmonary disorder such as asthma or COPD.

A suitable dose of an antibody or fragment thereof described herein,which dose is capable of treating or preventing a complement-associateddisorder in a subject, can depend on a variety of factors including,e.g., the age, sex, and weight of a subject to be treated and theparticular inhibitor compound used. For example, a different dose of awhole anti-C5a antibody may be required to treat a subject with RA ascompared to the dose of a C5a-binding Fab′ antibody fragment required totreat the same subject. Other factors affecting the dose administered tothe subject include, e.g., the type or severity of thecomplement-mediated disorder. For example, a subject having RA mayrequire administration of a different dosage of an anti-C5a antibodythan a subject with AMD. Other factors can include, e.g., other medicaldisorders concurrently or previously affecting the subject, the generalhealth of the subject, the genetic disposition of the subject, diet,time of administration, rate of excretion, drug combination, and anyother additional therapeutics that are administered to the subject. Itshould also be understood that a specific dosage and treatment regimenfor any particular subject will also depend upon the judgment of thetreating medical practitioner (e.g., doctor or nurse).

An antibody described herein can be administered as a fixed dose, or ina milligram per kilogram (mg/kg) dose. In some embodiments, the dose canalso be chosen to reduce or avoid production of antibodies or other hostimmune responses against one or more of the active antibodies in thecomposition. While in no way intended to be limiting, exemplary dosagesof an antibody, such as an anti-C5a antibody include, e.g., 1-1000μg/kg, 1-100 μg/kg, 0.5-50 μg/kg, 0.1-100 μg/kg, 0.5-25 μg/kg, 1-20μg/kg, and 1-10 μg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25mg/kg, 1-20 mg/kg, 0.100 mg/kg to 1 mg/kg, and 1-10 mg/kg. Exemplarydosages of an antibody or antigen-binding fragment thereof describedherein include, without limitation, 0.1 μg/kg, 0.5 μg/kg, 1.0 μg/kg, 2.0μg/kg, 4 μg/kg, and 8 μg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg,4 mg/kg, 8 mg/kg, and 20 mg/kg.

A pharmaceutical composition can include a therapeutically effectiveamount of an anti-C5a antibody or antigen-binding fragment thereofdescribed herein. Such effective amounts can be readily determined byone of ordinary skill in the art based, in part, on the effect of theadministered antibody, or the combinatorial effect of the antibody andone or more additional active agents, if more than one agent is used. Atherapeutically effective amount of an antibody or fragment thereofdescribed herein can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody (and one or more additional active agents) to elicit a desiredresponse in the individual, e.g., amelioration of at least one conditionparameter, e.g., amelioration of at least one symptom of thecomplement-mediated disorder. For example, a therapeutically effectiveamount of an anti-C5a antibody can inhibit (lessen the severity of oreliminate the occurrence of) and/or prevent a particular disorder,and/or any one of the symptoms of the particular disorder known in theart or described herein. A therapeutically effective amount is also onein which any toxic or detrimental effects of the composition areoutweighed by the therapeutically beneficial effects.

Suitable human doses of any of the antibodies or fragments thereofdescribed herein can further be evaluated in, e.g., Phase I doseescalation studies. See, e.g., van Gurp et al. (2008) Am JTransplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res13(2, part 1):523-531; and Hetherington et al. (2006) AntimicrobialAgents and Chemotherapy 50(10): 3499-3500.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose,” or similar terms used herein are intended to mean anamount of an agent (e.g., an anti-C5a antibody or an antigen-bindingfragment thereof) that will elicit the desired biological or medicalresponse (e.g., an improvement in one or more symptoms of acomplement-associated disorder). In some embodiments, a compositiondescribed herein contains a therapeutically effective amount of anantibody, or antigen-binding fragment thereof, which specifically bindsto a neo-epitope present in C5a. In some embodiments, the compositioncontains any of the antibodies or antigen-binding fragments thereofdescribed herein and one or more (e.g., two, three, four, five, six,seven, eight, nine, 10, or 11 or more) additional therapeutic agentssuch that the composition as a whole is therapeutically effective. Forexample, a composition can contain an anti-C5a antibody described hereinand an immunosuppressive agent, wherein the antibody and agent are eachat a concentration that when combined are therapeutically effective fortreating or preventing a complement-associated disorder (e.g., acomplement-associated inflammatory disorder such as COPD, asthma,sepsis, or RA) in a subject.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the complement-mediated disordersdescribed herein). Use of an anti-C5a antibody in an animal model of RAis exemplified in the working examples. These procedures can be used,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Anantibody or antigen-binding fragment thereof that exhibits a hightherapeutic index is preferred. While compositions that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue and tominimize potential damage to normal cells and, thereby, reduce sideeffects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch antibodies or antigen-binding fragments thereof lies generallywithin a range of circulating concentrations of the antibodies orfragments that include the ED₅₀ with little or no toxicity. The dosagemay vary within this range depending upon the dosage form employed andthe route of administration utilized. For an anti-C5a antibody describedherein, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the antibody which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography. In some embodiments, e.g., where local administration(e.g., to the eye or a joint) is desired, cell culture or animalmodeling can be used to determine a dose required to achieve atherapeutically effective concentration within the local site.

In some embodiments, the methods can be performed in conjunction withother therapies for complement-associated disorders. For example, thecomposition can be administered to a subject at the same time, prior to,or after, plasmapheresis, IVIG therapy, or plasma exchange. See, e.g.,Appel et al. (2005) J Am Soc Nephrol 16:1392-1404. In some embodiments,the composition can be administered to a subject at the same time, priorto, or after, a kidney transplant.

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human, a non-human primate (e.g., monkey, baboon, orchimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, arabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In someembodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a composition comprising an anti-C5a antibody).

The term “preventing” is art-recognized, and when used in relation to acondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of acomplement-associated disorder such as asthma includes, for example,reducing the extent or frequency of coughing, wheezing, or chest pain ina population of patients receiving a prophylactic treatment relative toan untreated control population, and/or delaying the occurrence ofcoughing or wheezing in a treated population versus an untreated controlpopulation, e.g., by a statistically and/or clinically significantamount.

As described above, the antibodies and biologically-active fragmentsdescribed herein can be used to treat a variety of complement-associateddisorders such as, but not limited to: rheumatoid arthritis (RA); lupusnephritis; ischemia-reperfusion injury; atypical hemolytic uremicsyndrome (aHUS); typical or infectious hemolytic uremic syndrome (tHUS);dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH);multiple sclerosis (MS); macular degeneration (e.g., age-related maculardegeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; sepsis; dermatomyositis; diabeticretinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneousfetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrentfetal loss; multiple sclerosis (MS); and traumatic brain injury. See,e.g., Holers (2008) Immunological Reviews 223:300-316 and Holers andThurman (2004) Molecular Immunology 41:147-152. In some embodiments, thecomplement-mediated disorder is a complement-mediated vascular disordersuch as, but not limited to, a cardiovascular disorder, myocarditis, acerebrovascular disorder, a peripheral (e.g., musculoskeletal) vasculardisorder, a renovascular disorder, a mesenteric/enteric vasculardisorder, revascularization to transplants and/or replants, vasculitis,Henoch-Schönlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, Takayasu's disease,capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy,thorasic-abdominal aortic aneurism, Kawasaki's disease (arteritis),venous gas embolus (VGE), and restenosis following stent placement,rotational atherectomy, and percutaneous transluminal coronaryangioplasty (PTCA). (See, e.g., U.S. patent application publication no.20070172483.) In some embodiments, the complement-associated disorder ismyasthenia gravis, cold-agglutinin disease (CAD), paroxysmal coldhemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmunehemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type Idiabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA),idiopathic thrombocytopenic purpura (ITP), Goodpasture syndrome,antiphospholipid syndrome (APS), Degos disease, and catastrophic APS(CAPS).

In some embodiments, an anti-C5a antibody or antigen-binding fragmentthereof described herein, alone or in combination with a secondanti-inflammatory agent, can be used to treat an inflammatory disordersuch as, but not limited to, RA (above), inflammatory bowel disease,sepsis (above), septic shock, acute lung injury, disseminatedintravascular coagulation (DIC), or Crohn's disease. In someembodiments, the second anti-inflammatory agent can be one selected fromthe group consisting of NSAIDs, corticosteroids, methotrexate,hydroxychloroquine, anti-TNF agents such as etanercept and infliximab, aB cell depleting agent such as rituximab, an interleukin-1 antagonist,or a T cell costimulatory blocking agent such as abatacept.

In some embodiments, the complement-associated disorder is acomplement-associated neurological disorder such as, but not limited to,amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease,and chronic inflammatory demyelinating neuropathy.

Complement-associated disorders also include complement-associatedpulmonary disorders such as, but not limited to, asthma, bronchitis, achronic obstructive pulmonary disease (COPD), an interstitial lungdisease, α-1 anti-trypsin deficiency, emphysema, bronchiectasis,bronchiolitis obliterans, alveolitis, sarcoidosis, pulmonary fibrosis,and collagen vascular disorders.

In the case of complement-associated hemolytic disorders such as PNH,CAD, and PCH, a medical practitioner will appreciate that C5 fragmentC5b (by way of the terminal complement complex) contributessignificantly to the pathogenesis of these disorders. See, e.g., Kaplan(2002) Curr Opin Investig Drugs 3(7):1017-23; Hill (2005) Clin AdvHematol Oncol 3(11):849-50; and Rother et al. (2007) NatureBiotechnology 25(11):1256-1488. Accordingly, a medical practitioner mayelect to administer one or more of the anti-C5a antibodies describedherein in conjunction with one or more additional therapies for thehemolytic disorder such as a complement inhibitor that preventsformation of the C5b-9 terminal complement complex. In some embodimentsof the methods described herein, the complement-associated disorder isnot a complement-associated hemolytic disorder. In some embodiments, ananti-C5a antibody or an antigen-binding fragment thereof is administeredto a subject to treat, prevent, or ameliorate at least one symptom of acomplement-associated inflammatory response (e.g., thecomplement-associated inflammatory response aspect of acomplement-associated disorder) in a subject. For example, an anti-C5aantibody described herein can be used to treat, prevent, and/orameliorate one or more symptoms associated with a complement-associatedinflammatory response such as graft rejection/graft-versus-host disease(GVHD), reperfusion injuries (e.g., following cardiopulmonary bypass ora tissue transplant), and tissue damage following other forms oftraumatic injury such as a burn (e.g., a severe burn), blunt trauma,spinal injury, or frostbite. See, e.g., Park et al. (1999) Anesth Analg99(1):42-48; Tofukuji et al. (1998) J Thorac Cardiovasc Surg116(6):1060-1068; Schmid et al. (1997) Shock 8(2):119-124; and Bless etal. (1999) Am J Physiol 276(1):L57-L63.

In some embodiments, an anti-C5a antibody or an antigen-binding fragmentthereof described herein can be administered to a subject as amonotherapy. Alternatively, as described above, the antibody or fragmentthereof can be administered to a subject as a combination therapy withanother treatment, e.g., another treatment for a complement-associateddisorder or a complement-associated inflammatory response. For example,the combination therapy can include administering to the subject (e.g.,a human patient) one or more additional agents (e.g., anti-coagulants,anti-hypertensives, or anti-inflammatory drugs (e.g., steroids)) thatprovide a therapeutic benefit to a subject who has, or is at risk ofdeveloping, sepsis. In another example, the combination therapy caninclude administering to the subject one or more additional agents(e.g., an anti-IgE antibody, an anti-IL-4 antibody, an anti-IL-5antibody, or an anti-histamine) that provide therapeutic benefit to asubject who has, is at risk of developing, or is suspected of having acomplement-associated pulmonary disorder such as COPD or asthma. In someembodiments, an anti-C5a antibody and the one or more additional activeagents are administered at the same time. In other embodiments, theanti-C5a antibody is administered first in time and the one or moreadditional active agents are administered second in time. In someembodiments, the one or more additional active agents are administeredfirst in time and the anti-C5a antibody is administered second in time.

An anti-C5a antibody or an antigen-binding fragment thereof describedherein can replace or augment a previously or currently administeredtherapy. For example, upon treating with an anti-C5a antibody orantigen-binding fragment thereof, administration of the one or moreadditional active agents can cease or diminish, e.g., be administered atlower levels. In some embodiments, administration of the previoustherapy can be maintained. In some embodiments, a previous therapy willbe maintained until the level of the anti-C5a antibody reaches a levelsufficient to provide a therapeutic effect. The two therapies can beadministered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in acomplement-associated disorder (e.g., sepsis, severe burn, RA, lupusnephritis, Goodpasture syndrome, or asthma), as defined herein, meansevaluating the subject for a change in a disease parameter, e.g., animprovement in one or more symptoms of a given disorder. The symptoms ofcomplement-associated disorders are well known in the art of medicine.In some embodiments, the evaluation is performed at least one (1) hour,e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after anadministration. The subject can be evaluated in one or more of thefollowing periods: prior to beginning of treatment; during thetreatment; or after one or more elements of the treatment have beenadministered. Evaluation can include evaluating the need for furthertreatment, e.g., evaluating whether a dosage, frequency ofadministration, or duration of treatment should be altered. It can alsoinclude evaluating the need to add or drop a selected therapeuticmodality, e.g., adding or dropping any of the treatments for acomplement-associated disorder described herein.

Therapeutic and Diagnostic Kits

The disclosure also features therapeutic and diagnostic kits containing,among other things, one or more of the anti-C5a antibodies, and/orantigen-binding fragments thereof, described herein. The therapeutickits can contain, e.g., a suitable means for delivery of the antibody orantigen-binding fragment to a subject. In some embodiments, the means issuitable for subcutaneous delivery of the antibody or antigen-bindingfragment thereof to the subject. The means can be, e.g., a syringe or anosmotic pump. That is, a therapeutic kit described herein can contain asyringe pre-filled with an anti-C5a antibody or antigen-binding fragmentthereof (e.g., a pen device containing the antibody or fragment)described herein or the kit can contain a pump (e.g., an osmotic pump)and one or more disposable cassettes configured for use with the pump,the cassettes pre-filled with an anti-C5a antibody or antigen-bindingfragment thereof described herein (e.g., prefilled with an aqueoussolution containing the anti-C5a antibody or antigen-binding fragmentthereof). In another example, the kit can contain a transscleral orimplantable delivery device (e.g., a plug) that is pre-filled with (orotherwise contains) a solution containing an anti-C5a antibody orantigen-binding fragment thereof described herein.

In some embodiments, the means for delivering an anti-C5a antibody orantigen-binding fragment thereof is a pen device for drug delivery.

In some embodiments, the means is suitable for intrapulmonary deliveryof the antibody or antigen-binding fragment thereof to a subject, e.g.,for use in treatment or prevention of a complement-associated pulmonarydisorder such as, but not limited to, COPD or asthma. Accordingly, themeans can be, e.g., an oral or nasal inhaler (see above). The inhalercan be, e.g., a metered dose inhaler (MDI), dry powder inhaler (DPI), ora nebulizer. Such a kit can also, optionally, include instructions foradministering (e.g., self-administration of) the anti-C5a antibody orantigen-binding fragment thereof to a subject.

The therapeutic kits can include, e.g., one or more additional activeagents for treating or preventing a complement-associated disorderand/or ameliorating a symptom thereof. For example, therapeutic kitsdesigned for use in treating or preventing a complement-associatedpulmonary disorder can include one or more additional active agentsincluding, but not limited to, another antibody therapeutic (e.g., ananti-IgE antibody, an anti-IL-4 antibody, or an anti-IL-5 antibody), asmall molecule anti-IgE inhibitor (e.g., montelukast sodium), asympathomimetic (e.g., albuterol), an antibiotic (e.g., tobramycin), adeoxyribonuclease (e.g., pulmozyme), an anticholinergic drug (e.g.,ipratropium bromide), a corticosteroid (e.g., dexamethasone), aβ-adrenoreceptor agonist, a leukotriene inhibitor (e.g., zileuton), a5-lipoxygenase inhibitor, a phosphodiesterase (PDE) inhibitor, a CD23antagonist, an IL-13 antagonist, a cytokine release inhibitor, ahistamine H1 receptor antagonist, an anti-histamine, ananti-inflammatory agent (e.g., cromolyn sodium or any otheranti-inflammatory agent known in the art or described herein), or ahistamine release inhibitor.

In some embodiments, the means can be suitable for intraocularadministration of an anti-C5a antibody, or an antigen-binding fragmentthereof, described herein to a subject in need thereof, e.g., a subjectafflicted with AMD or any other complement-associated ocular disorder.The means can be, e.g., a syringe, a trans-scleral patch, or even acontact lens containing the antibody or fragment. The means can, in someembodiments, be an eye dropper, wherein the anti-C5a antibody orantigen-binding fragment thereof is formulated for such administration.Such therapeutic kits can also include, e.g., one or more additionaltherapeutic agents for use in treating complement-associated disorder ofthe eye. The therapeutic agents can be, e.g., bevacizumab or the Fabfragment of bevacizumab, ranibizimab, both sold by RochePharmaceuticals, Inc., or pegaptanib sodium (Mucogen®; Pfizer, Inc.).Such a kit can also, optionally, include instructions for administeringthe anti-C5a antibody or antigen-binding fragment thereof to a subject.

In some embodiments, the means can be suitable for intraarticularadministration of an anti-C5a antibody, or antigen-binding fragmentthereof, described herein to a subject in need thereof, e.g., a subjectafflicted with RA. The means can be, e.g., a syringe or adouble-barreled syringe. See, e.g., U.S. Pat. Nos. 6,065,645 and6,698,622. A double-barreled syringe is useful for administering to ajoint two different compositions with only one injection. Two separatesyringes may be incorporated for use in administering the therapeuticwhile drawing off knee fluid for analysis (tapping) in a push-pullfashion. Additional therapeutic agents that can be administered with theanti-C5a antibodies or fragments in conjunction with the double-barreledsyringe, or which can otherwise be generally included in the therapeutickits described herein, include, e.g., NSAIDs, corticosteroids,methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept andinfliximab, a B cell depleting agent such as rituximab, an interleukin-1antagonist, or a T cell costimulatory blocking agent such as abatacept.Such a kit can also, optionally, include instructions for administeringthe anti-C5a antibody or antigen-binding fragment thereof to a subject.It will be appreciated that the disclosure embraces kits comprising oneor more of the anti-C5a antibodies described herein and one or moreanti-inflammatory agents selected from the group consisting of NSAIDs,corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents suchas etanercept and infliximab, a B cell depleting agent such asrituximab, an interleukin-1 antagonist, or a T cell costimulatoryblocking agent such as abatacept. The antibodies and agents can be,e.g., formulated separately or together. The kits can be used to treatan inflammatory condition such as RA, Crohn's disease, inflammatorybowel disease, or any other inflammatory disorder known in the art orrecited herein.

Also featured are diagnostic kits containing the anti-C5a antibodies orantigen-binding fragments thereof described herein. For example, thekits can contain a detectably-labeled form of an anti-C5a antibody(e.g., an anti-C5a antibody or an anti-mouse C5a antibody) describedherein for use in detecting or quantitating the amount of C5a in abiological sample. In some embodiments, the kits can contain isolatedC5a protein (e.g., one or both of human and mouse C5a protein) and/or acontrol sample comprising one or both of human and mouse C5a protein. Insome embodiments, the kit contains a multi-well plate coated with afirst anti-C5a antibody having a first specificity. The kit alsocontains a second anti-C5a antibody (e.g., a detectably-labeled secondanti-C5a antibody) having a second specificity. Such a kit is designedfor use in capturing, with the first antibody bound to the plate, C5aprotein (e.g., human C5a protein) in a sample (e.g., a biologicalsample) contacted to the plate and then detecting the captured C5aprotein using the second antibody. In some embodiments, diagnostic kitsinclude both an anti-mouse C5a antibody and an anti-human C5a antibodydescribed herein. In some embodiments, the diagnostic kits include ananti-C5a antibody that binds to both mouse C5a and human C5a.

The following examples are intended to illustrate, not limit, theinvention.

EXAMPLES Example 1 Immunization Methods

The presently described anti-C5a antibodies are humanized forms ofmurine antibodies generated under the following immunization protocol.Immunizations to raise antibodies against human desarginated C5a wereperformed on four mice including two mice of the strain DBA/2J and twomice of the strain A/J. These strains were selected because they carrythe allele Hc⁰, which makes them deficient in endogenous C5. Allimmunizations were repeated at 14 day intervals for a total of threeimmunizations. All animals received a subcutaneous booster immunizationof approximately 50 μg of purified C5a in 200 μl of adjuvant emulsionapproximately 14 days after the last immunization and 5 to 7 days beforeharvesting. Titering of serum from immunized mice, using an ELISA assay,showed that the mice exhibited a strong antibody response against thehuman desarginated C5a immunogen.

Example 2 Determining the Specificity of the Mouse Antibodies for HumanC5a

A subset of five mouse anti-human C5a Fabs that were representative ofneoepitope selective Fabs were converted to full length mouse IgG2aantibodies designated as—5an048ME, 5an101ME, 5an178ME, 5an179ME, and5an180ME. These antibodies were evaluated for specificity using BiolayerInterferometry on an Octet (ForteBio Inc.). (The amino acid sequences ofthe light chain and heavy chain CDR sets of each antibody, as defined byKabat, are set forth in Table 3.) Briefly, human C5a, human C5a des Arg,human full-length C5, or C5a paralogs human C3a and human C4a wereconjugated to biotin at a stoichiometry of <1(biotin):1 (antibody)through amine groups and immobilized on a streptavidin tip. Loaded tipswere then exposed to a solution containing 20 nM of anti-C5a IgGantibody. Each of the antibodies bound to C5a and desarginated C5a. Noneof the anti-C5a IgG antibodies bound to C3a or to C4a. However, 5an178MEand 5an179ME each bound to full-length human C5. A small amount ofbinding was observed between 5an048ME and full-length human C5. However,the binding of 5an048ME to C5 was much less than the binding observed toC5a.

These results confirmed that mouse anti-human C5a antibodies—5an048ME,5an101ME, and 5an180ME—bound to a neoepitope on C5a that was occluded innative, full-length C5 or generated after the cleavage of C5 intofragments C5a and C5b. The results also indicated that the threeantibodies were selective for human C5a as compared to paralogs C3a orC4a.

TABLE 3 Amino Acid Sequences for Five Murine Anti-Human C5a AntibodiesAb SIN: Description Amino Acid Sequence 5an048 151 V_(L) Amino AcidEIVLTQSPAIMSASPGEKVTMTCRASSSVSSS ME SequenceYLHWYQQKSGASPKLWIYSTSNLASGVPAR FSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGGGTKLEIKR 140 Light Chain CDR1 RASSSVSSSYLH 96 Light Chain CDR2STSNLAS 142 Light Chain CDR3 QQYSGYPLT 152 V_(H) Amino AcidEVRLQQSGPELVKPGASVRISCKASGYTFN Sequence DYYYMNWVKQSHGKSLEWIGYIFPKTGGTHYNQRFKGKATLTVDKSSSTAYMELRSLTS EDSAVYYCASGPFAYWGQGTLVTVSA 115 HeavyChain CDR1 DYYYMN 144 Heavy Chain CDR2 YIFPKTGGTHYNQRFKG 117 Heavy ChainCDR3 GPFAY 5an101 153 V_(L) Amino Acid DIVMTQSPASLAVSLGQRATISCRASESVDSME Sequence YGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC QQSNEDPYTFGGGTKLEIKR 20 Light Chain CDR1RASESVDSYGNSFMH 21 Light Chain CDR2 RASNLES 22 Light Chain CDR3QQSNEDPYT 154 V_(H) Amino Acid EVQLQQSGPELVKPGSSVKISCKASGYTFTD SequenceYSMDWVKQSHGKSLEWIGAINPNSGGTNY SQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCASSGSYDGYYAMDYWGQGTSVT VSS 28 Heavy Chain CDR1 DYSMD 67 HeavyChain CDR2 AINPNSGGTNYSQKFKD 30 Heavy Chain CDR3 SGSYDGYYAMDY 5an180 155V_(L) Amino Acid DIQMTQSPASLSASVGETVTITCRASENIYSY ME SequenceLAWYQQKQGKSPQLLVYNAKTLAEGVPSR FSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIKR 156 Light Chain CDR1 RASENIYSYLA 157 Light Chain CDR2NAKTLAE 158 Light Chain CDR3 QHHYGTPYT 159 V_(H) Amino AcidEVQLQQPGAEIVRPGASVKLSCRASGYTFT Sequence DYWMNWVKQRPGQGLEWIGTIDPSDSYTIYNQKFKGKATLTVDTSSTTAYIQLSSLTSED SAVYFCARGEDYDVSSYTMDYWGQGTSVT VSS 160Heavy Chain CDR1 DYWMN 161 Heavy Chain CDR2 TIDPSDSYTIYNQKFKG 162 HeavyChain CDR3 GEDYDVSSYTMDY 5an178 163 V_(L) Amino AcidEIVLTQSPASLAVSLGQRATISCSASESVEYF ME SequenceGTSLMQWYQQKPGQPPKLLIYAASNVESG VPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR 164 Light Chain CDR1 SASESVEYFGTSLMQ 165 LightChain CDR2 AASNVES 166 Light Chain CDR3 QQSRKVPWT 167 V_(H) Amino AcidEVKLVESGGGLVQPGGSRKLSCAASGFTFS Sequence DYGMVWVRQAPGKGLEWVAFISSGSSNIYYADTVKGRFTISRDNPKNTLFLQMNSLRSE DTAIYYCGRAFSFYYGYDYWGQGTTLTVSS 168 HeavyChain CDR1 DYGMV 169 Heavy Chain CDR2 FISSGSSNIYYADTVKG 170 Heavy ChainCDR3 AFSFYYGYDY 5an179 171 V_(L) Amino AcidDVVMTQTPLSLPVSLGDQASISCRSSQSLVH ME SequenceSNGNTYLHWYLQKPGQSPKLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKR 172 Light Chain CDR1 RSSQSLVHSNGNTYLH 173 LightChain CDR2 KVSNRFS 174 Light Chain CDR3 SQSTHVPLT 175 V_(H) Amino AcidEVQLQQSGPELVKPGASVRMSCKASGYTFT Sequence SYLIHWVKQKPGQGLEWIGYIYPFNDGTKNNENFKGKATLTSDKSSSTVYMEVSSLTSEDS AVYYCARSHGPHYYGGSYGYHFDYWGQG TTLTVSS 176Heavy Chain CDR1 SYLIH 177 Heavy Chain CDR2 YIYPFNDGTKNNENFKG 178 HeavyChain CDR3 SHGPHYYGGSYGYHFDY “SIN”in the Table refers to “SEQ ID NO.” *CDR amino acid sequence defined according to Kabat et al. (supra).

A series of sandwich assays were performed by Octet on the selectedsubset of mouse anti-human C5a IgG2a antibodies to determine the degreeof overlap of the C5a epitopes for each of the five representative IgG2aantibodies. Briefly, a first antibody was biotinylated and immobilizedon a streptavidin coated tip on the Octet platform. Next, human C5a wascaptured from a 20 nM solution on the immobilized antibody. The tipcarrying the antibody-C5a complex was then exposed to a solutioncontaining 20 nM of an unlabeled second anti-C5a IgG antibody. Theelicitation of an additional association profile in the sensogram wouldindicate that the two antibodies bound C5a simultaneously in a ternarycomplex and that the binding epitopes for the two antibodies werenon-overlapping. A failure to obtain a second association profile uponaddition of the second antibody would indicate that the two antibodiesbound C5a in a competitive manner, i.e., the epitope on C5a to which thesecond antibody bound was occluded after binding by the first antibody.In contrast to non-competitive binding, competitive binding does notnecessarily indicate that the first and second antibodies recognized thesame, or even overlapping, epitopes on human C5a. Using this approachthe binding sites of the five representative anti-C5a antibodies wereassigned to 4 distinct epitopes on human C5a (FIG. 1). Antibodies5an048ME, 5an180ME, and 5an101ME competed with each other. While 5an048and 5an180 also competed with the non-neoepitope selective 5an179ME,5an101ME did not, which indicated that 5an048ME and 5an180ME recognize aneoepitope that is different than the epitope recognized by 5an101ME. Inaddition, while the non-neoepitope selective antibody 5an178ME competedwith non-neoepitope selective 5an179ME, only the latter competed with5an180ME and 5an048ME, showing that 5an179ME and 5an178ME bind differentepitopes that are accessible both in C5 and C5a. The results alsoindicate that some combinations of the antibodies could be used insandwich-based assays to detect and/or quantify the amount of C5a in asample.

Example 3 Humanization of Select Mouse Anti-Human C5a Antibodies

The variable regions of two related mouse anti-human C5aantibodies—5an101ME and 5an185ME—were selected for humanization as fulllength IgG antibodies. Humanization of light and heavy chain variableregions was based on identifying individual framework regions from humanantibodies (with a preference given to germline v-genes) with a highdegree of sequence identity to the original murine parent antibody.Methods for identifying suitable candidate framework regions aredescribed in U.S. Pat. No. 7,393,648 to Rother and Wu. Definitions offramework (FW) and complementarity determining regions (CDRs) wereperformed according to methods described by Kabat, Chothia and IMGT®(International ImmunoGenetics Information System; France). Briefly,database queries were performed independently for both the light andheavy chain variable regions with a variety of antibody fragmentsincluding: intact murine variable region from FW1 through FW4, intactmurine variable regions excluding CDRs and all possible fragments ofmurine variable regions including one, two or three frameworks with orwithout their flanking CDRs. Human frameworks were selected from thiscandidate pool based on their overall sequence identity to the originalmurine antibodies and fragments thereof. Routine molecular biologicalmethods were employed to assemble small combinatorial libraries, of lessthan 10³ members, in which each set of murine CDRs were flanked by allpossible combinations of selected human frameworks. These humanizedantibodies were expressed as soluble Fabs and evaluated for binding todesarginated C5a using ELISA. Fabs that bound to C5a were then subjectedto DNA sequence analysis.

From these binders a subset of six humanized Fabs were reformatted asfull length IgGs (human IgG2 or human IgG2/G4). Additional humanizationwas performed in two antibodies (BNJ371 and BNJ381) by replacing murineresidues in CDR2 of the light chain with their corresponding humangermline amino acids. The amino acid sequences of the humanized anti-C5aantibodies—BNJ364, BNJ367, BNJ371, BNJ378, BNJ366, BNJ369, BNJ381, andBNJ383—are set forth in Table 2 above.

Example 4 Determining the Affinity of the Humanized Anti-Human C5aAntibodies for C5a

The humanized antibodies were subjected to BIAcore analysis to quantifytheir respective affinities for human C5a. See, e.g., Karlsson andLarsson (2004) Methods Mol Biot 248:389-415. Briefly, each of thehumanized antibodies were screened with 3-4 concentrations of human C5a(antigen) using a capture technique. The antibodies were captured by anAnti-Fc (human) directly immobilized on a CM5 sensor chip with variousconcentrations in the range from 0.6 nM to 5.9 nM of human C5a passedover the sensor chip surface. The surface was regenerated with 20 mMHCl, 0.02% P20 after each cycle to remove bound antibody and antigen.The data was evaluated using Biacore BIAevaluation software using a 1:1Langmuir Model Fit (Rmax:Global Fit; RI:Local Fit). Kinetics informationsuch as (k_(a): Association Rate constant), (k_(d):Dissociation Rateconstant) and K_(D) (Equilibrium Dissociation constant) was obtainedfrom the fit. The results of the analyses are set forth in Table 4.These experiments were for screening purposes with a minimal number ofanalyte concentrations (3 to 4) with 1 duplicate. Therefore, theapproximate kinetics values are reported in Table 4.

TABLE 4 Affinity Measurements for Select Humanized Anti-C5a AntibodiesAntibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Designation (×10⁶) (×10⁻⁴)(×10⁻¹²) χ² BNJ364 0.991 6.38 644 0.819 BNJ367 3.94 7.78 198 0.848BNJ371 2.38 28.2 1180 9.52 BNJ378 1.93 5.76 298 3.63 BNJ366 1.05 1.58150 1.23 BNJ369 4.19 2.23 53.1 0.642 BNJ381 2.57 2.09 81.5 1.93 BNJ3832.12 1.5 70.4 2.52

All of the humanized antibodies specifically bound to human C5a with aK_(D) less than 1.20 nanomolar. All of the antibodies with the exceptionof BNJ371 bound to human C5a with a K_(D) less than 1 nanomolar. Threeof the antibodies, BNJ369, BNJ381, and BNJ383 bound to human C5a with aK_(D) less than 100 picomolar.

Example 5 Anti-C5a Antibodies Inhibit C5a-Mediated Signaling In Vitro

An in vitro neutrophil activation assay was used to evaluate theactivity of the humanized antibodies. The assay is generally describedin, e.g., Paczkowski et al. (1999) Br J Pharmacol 128(7):1461-1466, andserves to quantitate the amount of myeloperoxidase (MPO) produced byneutrophils as a measure of neutrophil activation. Briefly,polymorphonuclear cells, the majority of which being neutrophils, wereisolated using density centrifugation (mono-poly resolving mediumcatalogue number: 91698049; MP Biochemicals; Solon, Ohio) from wholeblood from a healthy donor. The cells were washed once withphosphate-buffered saline (PBS) and the red blood cells (RBC) removedfrom the cell population by lysis in a hypotonic solution (ACK lysisbuffer catalogue number 10-548E; Lonza). After two more washes with PBS,the RBC-free cells were resuspended at a concentration of 4×10⁶ cells/mLin Hank's Balanced Salt Solution (HBSS; Mediatech, catalogue number:21-023-CV), which was supplemented with calcium and magnesium andfurther supplemented with 0.1% gelatin (Sigma Aldrich; St. Louis, Mo.)[hereinafter the assay buffer].

Cytochalasin B (Sigma Aldrich) was added to the cell suspension in anamount sufficient to reach a concentration of 10 μg/mL. The suspensionwas then incubated for 10 minutes at 37° C. 100 μL of cells was added towells of U-bottomed 96-well plates. The wells of the plate were groupedinto several different sets. Each of several of the different sets ofwells contained an anti-C5a antibody: each well of set 1 contained ahumanized antibody that binds to uncleaved, native C5, but not to freeC5a; each well of set 2 contained the BNJ367 humanized anti-C5aantibody; each well of set 3 contained the BNJ369 humanized anti-C5aantibody; each well of set 4 contained the BNJ371 humanized anti-C5aantibody; each well of set 5 contained the BNJ378 humanized anti-C5aantibody; each well of set 6 contained the BNJ381 humanized anti-C5aantibody; and each well of set 7 contained the BNJ383 humanized anti-C5aantibody. Each well of an eighth set of wells contained no antibody. Arange of antibody concentrations was evaluated in each set of wells, therange including 0.08 nM, 0.4 nM, 2 nM, and 10 nM antibody.

C5a (obtained from Complement Technologies, Inc.) was evaluated at aconcentration of 2 nM. A 10× working concentration of 20 nM was preparedin the aforementioned assay buffer and 20 μL was added to each well.After addition of C5a to the wells, the plate was incubated for 10minutes at 37° C. Following the incubation, 60 μL of PBS was added toeach well of the plate. The plates were subjected to centrifugation at1200 rpm (approximately 335×g) for 10 minutes at room temperature. 100μL of the supernatant from each well was transferred to thecorresponding well of a second plate. 25 μL of substrate (Sigma Aldrichcatalogue number T0440) was added to each well of the second plate andthe peroxidase reaction was allowed to develop for approximately two tofive minutes. The reaction was terminated by the addition of 25 μL of 1NHCl. The OD at 450 nM was recorded.

As shown in FIG. 2, all of the humanized anti-C5a antibodies inhibitedneutrophil activation in vitro. These results indicate that thehumanized anti-C5a antibodies described herein are potent inhibitors ofC5a-mediated signaling in vitro and support the conclusion by theinventors that the antibodies are useful for treating a variety ofcomplement-associated disorders (e.g., complement-associatedinflammatory disorders) in humans.

Example 6 Characterization of a Surrogate Mouse Anti-Mouse C5a Antibody

A series of sandwich assays were performed on a selected mouseanti-mouse C5a IgG antibody—5an195ME—to determine the specificity of theantibody for C5a. Briefly, the wells of an assay plate were coated withthe 5an195ME antibody. The plate was washed thoroughly to remove unboundantibody. Next, wells containing 5an195ME were contacted with mouse C5afor a time and under conditions sufficient to allow the antigen to bindto the antibody. Unbound protein was removed with washing. Following thewash step, the wells were further contacted with a solution containing asecond, biotinylated anti-C5a antibody. The wells were again washed toremove any unbound second antibody. The amount of binding of the secondantibody to the well was quantified using streptavidin-conjugatedhorseradish peroxidase (HRP). The amount of binding of the secondantibody was a function of the binding of C5a to 5an195ME.

In a parallel experiment, a set of 5an195ME-coated wells were incubatedwith full-length mouse C5 protein, rather than C5a. Following a washstep, the wells were contacted with a solution containing a secondantibody: a biotinylated anti-mouse C5 antibody. The amount of bindingof the second antibody, as a function of the amount of C5 bound by5an195ME, was quantified using the streptavidin-conjugated HRPconstruct. A lack of binding of the second antibody indicates that5an195ME does not bind to full-length mouse C5.

While 5an195ME bound to C5a in a dose-dependent manner, no bindingbetween the antibody and full-length mouse C5 was detected using thisassay. These results indicate that 5an195ME binds to a neo-epitopepresent in C5a.

The relative binding affinity of 5an195ME for mouse C5a was furtherquantified using BIAcore. The kinetics of 5an195ME were measured using acapture technique. The antibody was captured by an Anti-Fc (mouse)directly immobilized on a CM5 sensor chip with various concentrationsbetween and inclusive of 0.4 nM and 25 nM of mouse C5a passed over thesensor chip surface. Duplicates of each concentration were also run. Thesurface of the chip was regenerated with 10 mM glycine HCl pH 1.7 aftereach cycle to remove bound antibody and antigen. The data were evaluatedusing Biacore BIAevaluation software using a 1:1 Langmuir Model Fit(Rmax:Global Fit; RI:Local Fit). Kinetics information such as k_(a)(Association Rate constant), k_(d) (Dissociation Rate constant) andK_(D) (Equilibrium Dissociation constant) was obtained from the fit. Theresults of the kinetics analyses are shown in Table 5.

TABLE 5 Measured Kinetics of 5an195ME for Mouse C5a Parameter: k_(a)(1/Ms) k_(d) (1/s) K_(D) (M) χ² 5an195ME 8.47 × 10⁵ 1.27 × 10⁻³ 1.5 ×10⁻⁹ 1.17

These results indicate that the mouse anti-mouse C5a antibody is notonly specific for C5a, as compared to full-length mouse C5, but alsothat the antibody binds with high affinity to mouse C5a.

Example 7 Use of the Surrogate Anti-Mouse C5a Antibody 5an195ME in an RAAnimal Model

The 5an195ME anti-mouse C5a antibody was evaluated in a mouse model ofcollagen-induced arthritis. Male DBA/1LacJ mice (9 to 12 weeks old) wereimmunized by intradermal injection at the base of the tail with 300 μgof bovine type II collagen emulsified with equal volumes of Freund'scomplete adjuvant. The procedure was repeated two weeks after the firstimmunization. Mice were inspected daily to identify inflammation at aninitial knee joint. Once the initial inflammation was identified, micewere intraperitoneally administered three times/week the 5an195MEanti-mouse C5a antibody (40 mg/kg) or a control antibody (40 mg/kg). Thethickness of the initially inflamed joint (in mm) was measured daily today 12.

As shown in FIG. 3, 5an195ME reduced knee joint thickness as compared tothe control antibody. 5an195ME appeared to provide the benefit ofmaintaining a knee joint thickness below 4.5 mm.

In addition to evaluating the ability of the 5an195ME antibody to reduceswelling of the initially inflamed knee joint, the ability of the5an195ME anti-C5a antibody to prevent migration of inflammation to newjoints was also evaluated. The number of newly recruited joints wasmeasured daily from day 1 to day 12. The results of the experiment areset forth in Table 6.

TABLE 6 Efficacy of 5an195ME in RA Model Number of Number of Averagenumber Number joints newly of newly Treat- of inflamed inflamed jointsinflamed joints ment Mice on day 1 on day 12 per mouse Control Ab 6 7 122 5an195ME 6 7 2 0.3

As shown in Table 6, mice treated with 5an195ME had markedly fewer newlyinflamed joints as compared with control Ab treated animals by day 12.5an195ME-treated mice also had on average markedly fewer newly inflamedjoints.

The arthritis in the mice was also monitored and defined using aclinical score/arthritis index. Each limb was graded daily according toan established scoring system (0, normal joint; 1, mild/moderate visibleerythema and swelling; 2, severe erythema and swelling affecting anentire paw or joint; 3, deformed paw or joint with ankylosis.), with amaximum score of twenty-four per animal. See, e.g., Wang et al. (2000) JImmunol 164:4340-4347. As shown in FIG. 4, mice treated with theanti-mouse C5a antibody 5an195ME exhibited a marked reduction inclinical score (average score of less than 1), as compared to micetreated with the control antibody (average score above 6), over thecourse of the study.

In summary, these results indicate that the surrogate anti-mouse C5a iseffective in treating RA—both at an initial joint and the migration ofinflammation to secondary joints—in the mouse model of disease. Theresults also strongly suggest that a therapeutic anti-human C5aantibody, such as any of the humanized anti-C5a antibodies describedherein, is useful for treating humans with RA.

Example 8 Use of an Anti-C5a Antibody to Treat Rheumatoid Arthritis

A human patient is identified by a medical practitioner as havingrheumatoid arthritis in a single articulated joint. The patient isshortly thereafter administered intraarticularly or intraperitoneally acomposition containing a humanized anti-C5a antibody described herein inan amount sufficient to reduce C5a-mediated CSaR1 signaling locallywithin the joint space. The patient and medical practitioner observe asubstantial improvement in at least two known symptoms of rheumatoidarthritis following the treatment. The patient receives intravenouslyadministered “maintenance doses” of the antibody every month to preventreoccurrence of the symptoms, to prevent the progression of RA at thesingle joint, or to prevent the migration of RA symptoms to a secondjoint.

Example 9 Use of an Anti-C5a Antibody to Treat Sepsis

A human patient is identified by a medical practitioner as havingsepsis. The patient is shortly thereafter administered a compositioncontaining a humanized anti-C5a antibody described herein at a dose ofapproximately 600 to 900 mg by way of intravenous infusion. The patientand medical practitioner observe a substantial improvement in at leasttwo known symptoms of sepsis during the treatment. The patient receivesintravenously administered “maintenance doses” of the antibody every twoweeks until the patient leaves the hospital.

Example 10 Use of an Anti-C5a Antibody to Treat Complement-AssociatedPulmonary Inflammatory Disorders

A human patient is identified by a medical practitioner as having asevere form of COPD. Once every two weeks for four weeks the patient isadministered a composition containing a humanized anti-C5a antibody at adose of approximately 600 mg to 900 mg by intravenous infusion. Thepatient and medical practitioner observe a substantial improvement in atleast two known symptoms of COPD during the initial treatment. Forexample, the patient receiving the anti-C5a antibody has a reducedfrequency and/or severity of COPD-related exacerbations. The patientcontinues to receive intravenously administered “maintenance doses” ofthe antibody every two weeks to maintain the reduced frequency and/orseverity of COPD-related exacerbations.

A human patient is identified by a medical practitioner as having asevere form of asthma. The patient is prescribed a therapeutic,humanized anti-C5a antibody to be administered by way of an inhaler.During the next attack of bronchospasms, the patient self-administersthe anti-C5a antibody in an amount sufficient to reduce the C5a-mediatedinflammatory response in the lungs of the patient. The patient continuesto use the inhaler as needed to prevent or lessen the severity of asthmaattacks.

Example 11 Additional Anti-C5a Antibodies Identified from the ImmunizedMice

Several additional antibodies were obtained from the immunized mice (seeExample 1) and further identified by ELISA as capable of binding tohuman C5a. The additional antibodies include 15 unique light chain CDRsets (set forth in Table 7) and 14 unique heavy chain CDR sets (as setforth in Table 8).

TABLE 7 Amino Acid Sequences of Several Unique V_(L) and Kabat-definedLight Chain CDR sequences from Additional Murine Anti-human C5aAntibodies Ab SIN: Description Amino Acid Sequence* 5an110 83 V_(L)Amino Acid DIVMTQSQKFMSTSVGDRVSVTCKASQNVGT SequenceNVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPFTFGSGTKLEIKR 84 Light Chain CDR1 KASQNVGTNVA 85 Light Chain CDR2SASYRYS 86 Light Chain CDR3 QQYNSYPFT 5an177 87 V_(L) Amino AcidEIVLTQSPAIMSASPGEKVTMTCSASSSVSYMH SequenceWYQQKSGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKR 88 Light Chain CDR1 SASSSVSYMH 89 Light Chain CDR2 DTSKLAS90 Light Chain CDR3 QQWSSNPLT 5anG12 91 V_(L) Amino AcidQIVLTQSPAIMSASPGEKVTMTCSASSSISYMH SequenceWYQQKPGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIKR 92 Light Chain CDR1 SASSSISYMH 89 Light Chain CDR2 DTSKLAS93 Light Chain CDR3 HQRSSYPWT 5an052 94 V_(L) Amino AcidQIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYL SequenceYWYQQKPGSSPKLWIYSTSNLASGVPARFSGS GSGTSYSLTISTVEAEDAASYFCHQWSSYPPTFGGGTKLEIKR 95 Light Chain CDR1 SASSSVSSSYLY 96 Light Chain CDR2 STSNLAS97 Light Chain CDR3 HQWSSYPPT 5an107 98 V_(L) Amino AcidDIQMTQSPAPMLVSVGETVTITCRGSENIYSNL SequenceAWYQQKQGKSPQLLVYAATNLADGVPSRFSG SGSGTQYSLKINSLQSEDFGSYYCQHFWGTPRTFGGGTKLEIKR 99 Light Chain CDR1 RGSENIYSNLA 100 Light Chain CDR2AATNLAD 101 Light Chain CDR3 QHFWGTPRT 5anE11 102 V_(L) Amino AcidDIVMTQSQKFMSTSVGDRVSVTCKASQNVGT Sequence NVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYP WTFGGGTKLEIKR 84 Light Chain CDR1KASQNVGTNVA 85 Light Chain CDR2 SASYRYS 103 Light Chain CDR3 QQYNSYPWT5an054 104 V_(L) Amino Acid QIVLTQSPVIMSASPGEKVTMTCSASSSVSYM SequenceYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGS GSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGAGTKLELKR 105 Light Chain CDR1 SASSSVSYMY 106 Light Chain CDR2 DTSNLAS107 Light Chain CDR3 QQWSSYPPT 5anG10 141 V_(L) Amino AcidQIVLTQSPAIMSASPGEKVTMTCSASSSISYMH SequenceWYQQKPGTSPKRWIYDTSKLASGVPARFSGS GSGTSYSLTISSMEAEDAATYYCHQRRSYPWTFGGGTKLEIKR 92 Light Chain CDR1 SASSSISYMH 89 Light Chain CDR2 DTSKLAS108 Light Chain CDR3 HQRRSYPWT 5an188 109 V_(L) Amino AcidDIVLTQSPASLAVSLGQRATISCRASESVDSYG SequenceNSFMHWYQQKPGQPPKLLIYLASNLESGVPAR FSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPLTFGAGTKLELKR 20 Light Chain CDR1 RASESVDSYGNSFMH 110 Light Chain CDR2LASNLES 111 Light Chain CDR3 QQNNEDPLT 5an185 112 V_(L) Amino AcidDIVLTQSPASLAVSLGQRATISCRASESVDSYG SequenceNSFMHWYQQKPGQPPKLLIYRASNLESGIPAR FSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPLTFGAGTKLELKR 20 Light Chain CDR1 RASESVDSYGNSFMH 21 Light Chain CDR2RASNLES 113 Light Chain CDR3 QQSNEDPLT “SIN”in the Table refers to “SEQID NO.” *CDR amino acid sequences are defined according to Kabat et al.(supra).

TABLE 8 Amino Acid Sequences of Several Unique V_(H) and Kabat-definedHeavy Chain CDR sequences from Additional Murine Anti-human C5aAntibodies Ab SIN: Description Amino Acid Sequence* 5an110 114 V_(H)Amino Acid EVQLQQSGPELVKPGASVKISCKASGYTFSDY SequenceYYMNWVKKSHGKSLEWIGYIFPKTGGTNYS QRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 116 Heavy Chain CDR2YIFPKTGGTNYSQRFKG 117 Heavy Chain CDR3 GPFAY 5an177 118 V_(H) Amino AcidEVKLVESGGGLVKPGGSLKLSCAASGITFSSY Sequence YMAWVRQTPDKRLEWVATISSGGSYTYYPDNVKGRFTISRDNAKNTLYLQMSSLKSEDTAM YYCTRYYEDDAMDYWGQGTSVTVSS 119 HeavyChain CDR1 SYYMA 120 Heavy Chain CDR2 TISSGGSYTYYPDNVKG 121 Heavy ChainCDR3 YYEDDAMDY 5an055 122 V_(H) Amino AcidEVQLQQSGPELVKPGASVKISCKASGYTFSDY Sequence YYMNWVKKSHGKSLEWIGYIFPKTGGTNYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 123 Heavy Chain CDR2 YIFPKTGGTNYNQRFKG 117 Heavy Chain CDR3GPFAY 5an054 145 V_(H) Amino Acid EVQLQQSGPELVKPGASVKISCKASGYTFTDYSequence YYMNWVKQSHGKSLEWIGYIFPNTGGTTYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 124 Heavy Chain CDR2YIFPNTGGTTYNQRFKG 117 Heavy Chain CDR3 GPFAY 5an107 125 V_(H) Amino AcidEVKLVESGGGLVKPGGSLKLSCAASGYTFSS Sequence YYMAWVRQTPDKRLEWVATISSGGSYTYYRDNVKGRFTISRDNAKNTLYLQMSSLKSEDTA MYYCTRYFEDYPMDYWGQGTSVTVSS 119 HeavyChain CDR1 SYYMA 126 Heavy Chain CDR2 TISSGGSYTYYRDNVKG 127 Heavy ChainCDR3 YFEDYPMDY 5an111 128 V_(H) Amino AcidEVQLQQSGPELGKPGASGKISCKASGYTFTDY Sequence YYMNWVKQSHGKSLEWIGYIFPNTGGTSYNQRFKDKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 129 Heavy Chain CDR2 YIFPNTGGTSYNQRFKD 117 Heavy Chain CDR3GPFAY 5an183 130 V_(H) Amino Acid EVQLQQPGSVLVRPGATVKLSCKASGFTFTSSSequence WMHWAKQRPGQGLEWIGEIHTSGHTNYNEK FKGKATLTLDTSSSTAYVDISSLTSEDSAVYYCARGGLRRGYAMDYWGQGTSVTVSS 131 Heavy Chain CDR1 SSWMH 132 Heavy ChainCDR2 EIHTSGHTNYNEKFKG 133 Heavy Chain CDR3 GGLRRGYAMDY 5an185 134 V_(H)Amino Acid EVQPQQSGPELVKPGSSVKISCKASGYTFTDY SequenceSMDWVKQSHGKSLEWIGAIHLNTGYTNYNQ KFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGFYDGYSPMDYWGQGTSVTVSS 28 Heavy Chain CDR1 DYSMD 46 Heavy ChainCDR2 AIHLNTGYTNYNQKFKG 47 Heavy Chain CDR3 GFYDGYSPMDY 5an188 135 V_(H)Amino Acid EVQLQQSGAELVKPGTSVKLSCKASGYTFTS SequenceYWMHWVKQRPGQGLEYIGEIHPSSGHTNYH EKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARASLLRAYAMDYWGQGTSVTVSS 136 Heavy Chain CDR1 SYWMH 137 Heavy ChainCDR2 EIHPSSGHTNYHEKFKS 138 Heavy Chain CDR3 ASLLRAYAMDY “SIN”in theTable refers to “SEQ ID NO.” *CDR amino acid sequences are definedaccording to Kabat et al. (supra).

V_(L) and V_(H) pairings giving rise to the 5an177ME, 5an054ME,5an110ME, 5an188ME, 5an185ME, and 5an107ME antibodies are evident fromTables 7 and 8. Additional exemplary pairings of the heavy chain andlight chain variable regions and/or CDR sets are set forth in Table 9below.

TABLE 9 Amino Acid Sequences for Additional Mouse Anti-Human C5aAntibodies CDR Sets Ab SIN: Description Amino Acid Sequence* 5an047 139V_(L) Amino Acid EIVLTQSPAIMSASPGEKVTMTCRASSSVSSSY SequenceLHWYQQKSGASPKLWIYSTSNLASGVPARFS GSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELKR 140 Light Chain CDR1 RASSSVSSSYLH 96 Light Chain CDR2STSNLAS 142 Light Chain CDR3 QQYSGYPLT 143 V_(H) Amino AcidEVQLQQSGPELVKPGASVRISCKASGYTFSDY Sequence YYMNWVKKSHGKSLEWIGYIFPKTGGTHYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 144 Heavy Chain CDR2 YIFPKTGGTHYNQRFKG 117 Heavy Chain CDR3GPFAY 5an181 139 V_(L) Amino Acid EIVLTQSPAIMSASPGEKVTMTCRASSSVSSSYSequence LHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYP LTFGAGTKLELKR 140 Light Chain CDR1RASSSVSSSYLH 96 Light Chain CDR2 STSNLAS 142 Light Chain CDR3 QQYSGYPLT122 V_(H) Amino Acid EVQLQQSGPELVKPGASVKISCKASGYTFSDY SequenceYYMNWVKKSHGKSLEWIGYIFPKTGGTNYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 123 Heavy Chain CDR2YIFPKTGGTNYNQRFKG 117 Heavy Chain CDR3 GPFAY 5an109 146 V_(L) Amino AcidEIVLTQSPAIMSASPGEKVTMTCSASSSVSYM SequenceYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGS GSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGGTKLEIKR 105 Light Chain CDR1 SASSSVSYMY 106 Light Chain CDR2DTSNLAS 107 Light Chain CDR3 QQWSSYPPT 122 V_(H) Amino AcidEVQLQQSGPELVKPGASVKISCKASGYTFSDY Sequence YYMNWVKKSHGKSLEWIGYIFPKTGGTNYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 123 Heavy Chain CDR2 YIFPKTGGTNYNQRFKG 117 Heavy Chain CDR3GPFAY 5anG10 141 V_(L) Amino Acid QIVLTQSPAIMSASPGEKVTMTCSASSSISYMSequence HWYQQKPGTSPKRWIYDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQRRSYPWTFGGGTKLEIKR 92 Light Chain CDR1 SASSSISYMH 89 Light Chain CDR2 DTSKLAS108 Light Chain CDR3 HQRRSYPWT 147 V_(H) Amino AcidEVQLQQSGPELVKPGASVRISCKASGYTFND Sequence YYYMNWVKQSHGKSLEWIGYIFPKTGGTHYNQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 144 Heavy Chain CDR2 YIFPKTGGTHYNQRFKG 117 Heavy Chain CDR3GPFAY 5an053 148 V_(L) Amino Acid EIVLTQSPVIMSASPGEKVTMICSASSSISYMHSequence WYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISIMEAEDAATYYCHQRSSYPWT FGGGTKLEIKR 92 Light Chain CDR1SASSSISYMH 89 Light Chain CDR2 DTSKLAS 93 Light Chain CDR3 HQRSSYPWT 149V_(H) Amino Acid EVQMQQSGPELVKPGASVKISCKASGYTFSD SequenceYYYMNWVKKSHGKSLEWIGYIFPKTGGTNY NQRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 123 Heavy Chain CDR2YIFPKTGGTNYNQRFKG 117 Heavy Chain CDR3 GPFAY 5anG12 91 V_(L) Amino AcidQIVLTQSPAIMSASPGEKVTMTCSASSSISYM SequenceHWYQQKPGTSPKRWIYDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIKR 92 Light Chain CDR1 SASSSISYMH 89 Light Chain CDR2 DTSKLAS93 Light Chain CDR3 HQRSSYPWT 147 V_(H) Amino AcidEVQLQQSGPELVKPGASVRISCKASGYTFND Sequence YYYMNWVKQSHGKSLEWIGYIFPKTGGTHYNQRFKGKATLTVDKSSSTAYMELRSLTSEDS AVYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 144 Heavy Chain CDR2 YIFPKTGGTHYNQRFKG 117 Heavy Chain CDR3GPFAY 5an052 94 V_(L) Amino Acid QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYSequence LYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSYP PTFGGGTKLEIKR 95 Light Chain CDR1SASSSVSSSYLY 96 Light Chain CDR2 STSNLAS 97 Light Chain CDR3 HQWSSYPPT147 V_(H) Amino Acid EVQLQQSGPELVKPGASVRISCKASGYTFND SequenceYYYMNWVKQSHGKSLEWIGYIFPKTGGTHY NQRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 144 Heavy Chain CDR2YIFPKTGGTHYNQRFKG 117 Heavy Chain CDR3 GPFAY 5an111 102 V_(L) Amino AcidDIVMTQSQKFMSTSVGDRVSVTCKASQNVGT Sequence NVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSY PWTFGGGTKLEIKR 84 Light Chain CDR1KASQNVGTNVA 85 Light Chain CDR2 SASYRYS 103 Light Chain CDR3 QQYNSYPWT128 V_(H) Amino Acid EVQLQQSGPELGKPGASGKISCKASGYTFTDY SequenceYYMNWVKQSHGKSLEWIGYIFPNTGGTSYN QRFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 129 Heavy Chain CDR2YIFPNTGGTSYNQRFKD 117 Heavy Chain CDR3 GPFAY 5an055 150 V_(L) Amino AcidEIVLTQSPAIMSASPGEKVTLTCSASSSVSSSY SequenceLYWYQQKPGSSPKLWIYSTSNLASGVPARFS GSGSGTSYSLTISSMEAEDAASYFCHQWSSYPPTFGGGTKLEIKR 95 Light Chain CDR1 SASSSVSSSYLY 96 Light Chain CDR2STSNLAS 97 Light Chain CDR3 HQWSSYPPT 122 V_(H) Amino AcidEVQLQQSGPELVKPGASVKISCKASGYTFSDY Sequence YYMNWVKKSHGKSLEWIGYIFPKTGGTNYNQRFKGKATLTVDKSSSTAYMELRSLTSEDSA VYYCASGPFAYWGQGTLVTVSA 115 Heavy ChainCDR1 DYYYMN 123 Heavy Chain CDR2 YIFPKTGGTNYNQRFKG 117 Heavy Chain CDR3GPFAY 5anE11 102 V_(L) Amino Acid DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTSequence NVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSY PWTFGGGTKLEIKR 84 Light Chain CDR1KASQNVGTNVA 85 Light Chain CDR2 SASYRYS 103 Light Chain CDR3 QQYNSYPWT143 V_(H) Amino Acid EVQLQQSGPELVKPGASVRISCKASGYTFSDY SequenceYYMNWVKKSHGKSLEWIGYIFPKTGGTHYN QRFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASGPFAYWGQGTLVTVSA 115 Heavy Chain CDR1 DYYYMN 144 Heavy Chain CDR2YIFPKTGGTHYNQRFKG 117 Heavy Chain CDR3 GPFAY 5an183 112 V_(L) Amino AcidDIVLTQSPASLAVSLGQRATISCRASESVDSY SequenceGNSFMHWYQQKPGQPPKLLIYRASNLESGIP ARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPLTFGAGTKLELKR 20 Light Chain CDR1 RASESVDSYGNSFMH 21 Light ChainCDR2 RASNLES 113 Light Chain CDR3 QQSNEDPLT 130 V_(H) Amino AcidEVQLQQPGSVLVRPGATVKLSCKASGFTFTSS Sequence WMHWAKQRPGQGLEWIGEIHTSGHTNYNEKFKGKATLTLDTSSSTAYVDISSLTSEDSAVYY CARGGLRRGYAMDYWGQGTSVTVSS 131 HeavyChain CDR1 SSWMH 132 Heavy Chain CDR2 EIHTSGHTNYNEKFKG 133 Heavy ChainCDR3 GGLRRGYAMDY “SIN”in the Table refers to “SEQ ID NO.” *CDR aminoacid sequences are defined according to Kabat et al. (supra).

Example 12 Additional Humanized Anti-Human C5a Antibodies

Several additional humanized anti-C5a antibody heavy chain variableregions were generated, all of which when paired with a common lightchain variable region (the light chain having the amino acid sequencedepicted in SEQ ID NO:16) bound to human C5a with a K_(D) of less than 1nM as determined by Biacore analysis (see above for methodology). All ofthese additional humanized antibodies bound specifically to human C5a,but did not bind to native, fully-folded human C5, C4a, or C3a asdetermined by Octet analysis (see above for methodology). The additionalhumanized antibodies contained: (i) a heavy chain variable regionframework region 1 containing one of the following amino acid sequences:QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:68) orQVQLVQSGSELKKPGASVKVSCKASGYTFT (SEQ ID NO:69); (ii) a heavy chainvariable region framework region 2 containing one of the following aminoacid sequences: WVRQAPGQGLEWMG (SEQ ID NO:70) or WVRQASGKGLEWVG (SEQ IDNO:71); (iii) a heavy chain variable region framework region 3containing one of the following amino acid sequences:RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:72);RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:73); orRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR (SEQ ID NO:74); and a heavy chainvariable region framework region 4 containing the following amino acidsequence: WGQGTTVTVSS (SEQ ID NO:75). Exemplary additional humanizedheavy chain variable regions comprising one or more of the additionalhumanized framework sets described in this section are set forth in FIG.5.

Example 13 Use of an Anti-Human C5a Antibody in a Mouse NeutropeniaModel

A murine model of C5a-neutropenia was utilized to evaluate the efficacyof an anti-human free C5a antibody in vivo. To induce neutropenia,purified, native human C5a (hC5a) was administered by way of intravenoustail vein injection to Balb/c mice. The number of circulatingneutrophils was evaluated up to five minutes after administration ofhC5a.

Administration of 300 μg/kg of hC5a was consistently found to induceneutrophil activation as measured by the myeloperoxidase (MPO) releaseassay (see Example 5 for use with serum as compared to cell culturesupernatant, supra) and neutropenia (a reduction in the number ofcirculating neutrophils). In addition, plasma levels of hC5a and thehuman anti-C5a antibody BNJ383 (when administered to the mice, infra)were also measured to establish the pharmacodynamic response (seebelow).

Peripheral blood neutrophil counts were examined before challenge withhC5a or vehicle control. Compared to sham-treated control mice(1.37±0.09×10⁶/mL), neutrophil counts in mice treated with anti-humanfree C5a antibody (1.32±0.13×10⁶ per mL at 24 mg/kg; P>0.05) or isotypecontrol mAb (1.31±0.10×10⁶ per mL at 24 mg/kg; P>0.05) remained thesame. These results indicated that antibody alone did not induce changesin circulating neutrophil counts.

To evaluate the efficacy of an anti-C5a antibody to inhibit hC5a-inducedneutropenia in mice, different dosages (24 mg/kg, 12 mg/kg, 6 mg/kg, and3 mg/kg) of the anti-human C5a antibody BNJ383 were administered toBalb/c mice 24 hours prior to hC5a injection. Administration of theanti-C5a antibody 24 hours ahead of hC5a allowed for thepharmacodynamics properties of the antibody to be studied during theβ-phase of antibody elimination from the mice.

As shown in FIG. 6, neutrophil counts after treatment are expressed as apercentage of “baseline” (where the count at time 0 equals 100%). Insham-treated control mice, the neutrophil counts were 79.02±5.71%,67.42±3.23%, and 59.54±2.11% of baseline at 1, 3 and 5 minutes post hC5aadministration, respectively. The isotype control antibody-treated micedemonstrated a significant reduction (P<0.01) in the neutrophil count to6.76±0.81% at 1 minute, 6.68±0.81% at 3 minutes, and 8.29±0.79% at 5minutes after intravenous injection of hC5a. The anti-C5a antibodyexhibited a dose-dependent effect on hC5a-induced neutropenia. At thehighest dose, 24 mg/kg, the anti-C5a antibody completely blocked theneutropenia. Neutrophil counts were 70.35±8.64% at 1 minute, 63.35±6.08%at 3 minutes, and 59.65±6.51% at 5 minutes, which was comparable to theneutrophil levels in sham-treated control animals at the same timepoints. The lower doses of 12 mg/kg or 6 mg/kg of the anti-C5a antibodyalso significantly inhibited neutrophil depletion (12 mg/kg: 42.61±5.12%at 1 minute, 45.33±8.29% at 3 minutes, and 41.02±7.08% at 5 minutes,P<0.01; 6 mg/kg: 18.00±3.8 at 1 minute, 26.20±4.44% at 3 minutes, and28.03±4.51% at 5 minutes, P<0.05) following administration of hC5a, ascompared to the isotype control antibody group (6.76±0.81% at 1 minute,6.68±0.81% at 3 minutes, and 8.29±0.79% at 5 minutes). The isotypecontrol antibody is an antibody that binds anthrax protective antigen 63and contains a human IgG2/4 isotype Fc region. Administration of thelowest dose of the anti-C5a antibody (3 mg/kg) did not significantlyreduce neutropenia (6.28±0.88% at 1 minute, 6.71±2.14% at 3 minutes, and8.75±2.98% at 5 minutes, P>0.05). See FIG. 6.

Anti-Human C5a Antibody Inhibits hC5a-Induced MPO Release In Vivo

As discussed above, human C5a activates neutrophils throughcross-reactive binding of the mouse C5a receptor. Myeloperoxidase (MPO)release is a consequence of neutrophil activation through C5a binding toC5aR. See Darren et al. (2004) Mol Pharm 65(4):868-879. Intravenousinjection of recombinant human C5a into the mouse can induce neutropeniaand activate neutrophils in circulation. The ability of an anti-humanfree C5a antibody to inhibit MPO release due to neutrophil activation invivo was evaluated using the anti-C5a antibody to bind free hC5a andprevent binding to the murine C5aR.

An in vivo experiment (in which human C5a was administered to mice) wasperformed as described above. The level of plasma MPO at time 0 was79.25±22.88 ng/mL in the sham-treated control group. Five minutes afterintravenous injection of vehicle buffer, MPO levels were notsignificantly changed (77.46±21.21 ng/mL, P>0.05). Prior to intravenousinjection of hC5a, MPO levels in isotype control antibody-treated(75.17±14.66 ng/mL) or anti-C5a antibody-treated animals (87.57±14.86ng/mL) were comparable with the levels observed in the sham-treatedcontrol animals. After intravenous injection of hC5a, MPO levels at alldoses of C5a were raised and remained at significantly higher levels at5 minutes (FIG. 7).

When compared to isotype control antibody-treated animals (221.00±51.02ng/mL), the anti-hC5a antibody-treated animals showed dose-dependentreduction of MPO levels (114.83±23.26 ng/mL, P<0.05, in 24 mg/kg cohort;104.80±29.83 ng/mL, P<0.05, in 12 mg/kg cohort; and 126.90±36.40 ng/mL,P=0.08, in 6 mg/kg cohort). The MPO levels of low dose (3 mg/kg)anti-C5a antibody-treated animals (176.55±23.05 ng/mL) were notsignificantly different from isotype control antibody-treated animals(P>0.05).

An Anti-C5a Antibody Reduces Circulating hC5a Levels in Mice

As noted above, C5a is a potent inflammatory peptide with severalbiological functions. These above studies demonstrated that human C5across-reacts with murine C5aR on neutrophils since intravenous injectionof recombinant human C5a can induce neutropenia. While this disclosureis in no way limited by any particular theory or mechanism of action,the anti-C5a antibody may be inhibiting human C5a-induced neutropenia byforming complexes with hC5a and preventing hC5a from binding to themurine C5aR expressed on the cell surface. hC5a levels were measured inthe plasma of mice before and 1, 3, and 5 minutes after intravenousadministration of hC5a to confirm that the effects of the anti-hC5aantibody in vivo were due to its binding-dependent inhibition of hC5a.

An experiment was performed as described above using themouse-hC5a-induced neutropenia model. hC5a was not detected in plasma inany group of mice prior to administration of hC5a at time 0, using anenzyme-linked immunosorbent assay (ELISA). The level of hC5a in plasma,however, increased to a peak at 1 minute (7783.50±327.73 ng/mL), thenreduced to 4788.38±260.51 ng/mL at 3 minutes, and then to 3855.75±298.99ng/mL at 5 minutes following intravenous injection of hC5a (in isotypecontrol mAb-treated mouse). Compared to control antibody-treated mice,the level of hC5a in mice treated with 24 mg/kg of the anti-C5a antibodyexhibited a 43, 30, and 23-fold decline at 1 minute (178.4±14.14 ng/mL),3 minutes (158.4±10.43 ng/mL), and 5 minutes (167.2±15.61 ng/mL),respectively. The level of hC5a in plasma from the 12 mg/kg and 6 mg/kgcohorts were 235.00±22.33 and 609.20±78.75 ng/mL at 1 minute,210.80±19.59 and 527.60±52.25 ng/mL at 3 minutes, 192.20±7.40 and505.00±45.96 ng/mL at 5 minutes, respectively. The anti-C5a-treated miceexhibited significantly reduced hC5a levels in a dose-dependent mannerduring neutropenia following intravenous injection of hC5a (P<0.001).Although the mice receiving the lowest dose of anti-C5a antibody (3mg/kg) were not spared from hC5a-induced neutropenia, the micenonetheless had a significant reduction in plasma hC5a (3130.40±433.58ng/mL at 1 minute; 1932.00±268.92 ng/mL at 3 minutes; 1593.00±169.68ng/mL at 5 minutes) as compared to plasma hC5a levels found in isotypecontrol antibody-treated mice (P<0.05). See FIG. 8. These data indicatedthat administration of anti-C5a antibody to mice significantly decreasedthe concentration of free hC5a in plasma, resulting in greatlyameliorated hC5a-induced neutropenia.

Taken together, these results presented in this section indicated thatan anti-human C5a antibody described herein can inhibit the biologicaleffect of human C5a in an in vivo disease setting and provide strongevidence that the antibodies (and antigen-binding fragments thereof) areuseful in, among other things, treating or preventingcomplement-associated disorders such as any of those recited herein.

Example 14 Anti-Human C5a Antibody Crossreacts with C5a from Non-HumanPrimates

Several humanized anti-hC5a antibodies were tested for their ability tocrossreact with C5a from one or more non-human mammalian species. Asnoted above, the benefits of such an anti-C5a antibody are numerous,e.g., the ability of a research or medical practitioner to model theefficacy of a therapeutic anti-C5a antibody in a non-human disease modelprior to administering the antibody to humans. Testing in non-humanmammals can also allow for determination or approximation of theappropriate dosage of an anti-C5a antibody required for efficacy inhumans.

Briefly, BNJ369, BNJ366, BNJ364, and BNJ383 (described above) wereevaluated to determine whether they could co-immunoprecipitate C5aprotein in the activated serum from several non-human primates includingbaboon, rhesus macaque, and cynomolgus macaque. The serum was activatedby addition of zymosan. Following an overnight incubation of eachantibody with activated serum, the antibodies were separated from thesolution phase using protein A-conjugated agarose beads. The beads werewashed thoroughly and then boiled in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffercontaining β-mercaptoethanol. The boiled samples were then subjected toSDS-PAGE. Non-human primate C5a was detected by western blot using thecommercially-available anti-C5a neoepitope antibody #2942 (Abcam,Cambridge, Mass.). Each of the antibodies tested was capable ofimmunoprecipitating C5a (or C5a desarg) from baboon, rhesus macaque, andcynomolgus macaque indicating that the antibody is crossreactive withC5a from these species as well as with human C5a.

The determination of binding affinity parameters to cynomolgus macaqueC5a was determined as described above. Briefly, the BNJ383 antibody wasscreened against 3-4 concentrations of recombinant cynomolgus macaqueC5a (antigen) using a capture technique as described above. The antibodywas captured by an Anti-Fc (human) directly immobilized on a CM5 sensorchip with various concentrations in the range from 0.6 nM to 5.9 nM ofcynomolgus C5a passed over the sensor chip surface. The surface wasregenerated with 20 mM HCl, 0.02% surfactant P20 (Biacore) after eachcycle to remove bound antibody and antigen. The data was evaluated usingBiacore BIAevaluation software using a 1:1 Langmuir Model Fit(Rmax:Global Fit; RI:Local Fit). These experiments were for screeningpurposes with a minimal number of analyte concentrations (3 to 4) with 1duplicate. The approximate K_(D) of the antibody for cynomolgus macaqueC5a is 3.3 nM. See Table 10.

TABLE 10 Affinity Determination for non-Human C5a Proteins k_(a) (1/Ms)k_(d) (1/s) K_(D)(M) Species (×10⁶) (×10⁻⁴) (×10⁻¹²) χ² Human 0.77 8.32 108 1.23 Cynomolgus 1.28 42.3 3300 1.45 macaque Mouse 2.8 10.6  379*2.38 *This is only an approximation of the K_(D) based on the quality ofthe curve fit.

The BNJ383 antibody was also screened against 3-4 concentrations ofrecombinant mouse C5a (antigen) using a capture technique as describedabove to determine its affinity for mouse protein. The antibody wascaptured, as described above, by an anti-Fc (human) directly immobilizedon a CM5 sensor chip with various concentrations in the range from 0.6nM to 5.9 nM of mouse C5a passed over the sensor chip surface. Thesurface was regenerated with 20 mM HCl, 0.02% P20 after each cycle toremove bound antibody and antigen. The data were evaluated using BiacoreBIAevaluation software using a 1:1 Langmuir Model Fit (Rmax:Global Fit;RI:Local Fit).

The above results indicated that several of the humanized anti-hC5aantibodies described herein are crossreactive with C5a from severalnon-human primate species including cynomolgus macaque, rhesus macaque,and baboon. The BNJ383 antibody, e.g., also crossreacts with mouse C5a.Furthermore, the results described in this section indicate that ananti-human C5a antibody, such as BNJ383, is useful not only in clinicalapplications for treating complement-associated disorders, but also in avariety of pre-clinical applications in non-human mammals, which arenecessary for, or supportive of, approval of clinical use in humans.

Example 15 Competition for Binding to C5a

An experiment was performed to evaluate the binding of an anti-C5aantibody described herein, BNJ383, in the presence of potentiallycompetitive antigens. Briefly, ruthenium-labeled BNJ383 (250 pM) wasincubated for two hours at room temperature with 1 nM biotinylated C5a,along with various concentrations (e.g., 400, 133, 44.4, 14.8, 4.9, 1.6,and 0.5 nM) of one of the following: (a) human C5a desarg protein inphosphate-buffered saline, (b) human plasma, (c) cynomolgus macaqueplasma, (d) Balb/C (mouse) plasma, or (e) DBA/2J (mouse) plasma. Withrespect to the plasma components (b), (c), (d), and (e), theconcentration refers to the approximate final concentration of C5antigen in the incubation mixture.

Following the incubation period, the samples were contacted torespective individual wells of a streptavidin-coated assay plate underconditions that allowed for the binding of biotinylated C5a to thestreptavidin in the wells of the plate. The wells were washed thoroughlyto remove unbound material. The amount of binding of BNJ383 to C5a inthe presence of competitor was determined by detecting the amount ofsignal produced from the detectable ruthenium label. The results areshown in FIG. 9.

Whereas human C5a desarg was an effective competitor, there wasvirtually no competition observed in the presence of mouse serum (17%reduction in detectable signal observed at approximately a 400:1 ratioof Balb/C mouse plasma-derived C5 to biotinylated human C5a and 25%reduction in detectable signal observed at approximately a 400:1 ratioof DBA2/J plasma-derived C5 to biotinylated human C5a). No change in thelevel of binding of BNJ383 to biotinylated human C5a was observed at upto approximately a 15:1 ratio of human or cynomolgus macaqueplasma-derived C5 to biotinylated human C5a.

As noted above, while the disclosure is in no way limited to anyparticular theory or mechanism of action, the inventors hypothesize thatthe anti-C5a antibody may bind to a subpopulation of uncleaved,processed C5 (e.g., plasma C5) constituting less than 10% of the totalpopulation of full length C5 in a sample (e.g., a plasma sample), whichsubpopulation is in whole or in part denatured such that an otherwiseoccluded C5a neoepitope, to which the anti-C5a antibody or fragmentbinds, is exposed. Thus, it is believed that the antibody does not bindto a fully functional and/or fully functional species of C5 and thusdoes not truly bind to uncleaved, native C5. Human plasma is at least anapproximately 30 to 100-fold weaker competitor for binding tobiotinylated C5a than human C5a desarg.

Notwithstanding these considerations, these results further indicatethat anti-human C5a antibodies described herein, such as BNJ383,preferentially bind to free human C5a even in the presence of up toapproximately 20-fold excess of uncleaved, but not necessarily entirelynative, plasma-derived human C5 protein.

Example 16 Effect of Anti-C5a Antibody on AP and CP Activity In Vitro

An experiment was performed to evaluate the effect of an anti-C5aantibody described herein (BNJ383) on alternative pathway (AP)complement activity in vitro using pooled normal human serum (PNHS). Theexperiment utilized the Wieslab® Alternative Pathway Complement Kit(Wieslab® COMPL AP330, Euro-Diagnostica, Sweden) and the associatedprotocol was followed with only routine optimization well within thepurview of one of ordinary skill in the art. Briefly aliquots of thePNHS were incubated in wells of a lipopolysaccharide-coated plate forone hour (at 37° C.) along with various concentrations (0.778, 0.389,0.194, 0.097, 0.049, 0.024, 0.012, 0.006, 0.003, and 0.002 μM) of ananti-hC5 antibody or an anti-hC5a antibody (BNJ383). The anti-C5antibody inhibits the cleavage of human C5 into fragments C5a and C5b.As a negative control, several wells were incubated with PNHS under thesame conditions, but in the absence of anti-hC5 antibody or anti-hC5aantibody.

Following the incubation, the wells were washed thoroughly withkit-supplied 1× wash buffer. The level of alternative pathway complementactivation was measured by absorbance at 405 nm, following contact ofeach well with a kit-supplied enzyme conjugate (an anti-C5b-9 antibodyconjugated to alkaline phosphatase) and fluorogenic substrate (which isoperated upon by the enzyme) and incubation for 30 minutes at roomtemperature. The results are shown in FIG. 10.

While the anti-C5 antibody inhibited alternative pathway complementactivity completely at concentrations greater than 0.1 μM, the anti-hC5aantibody did not significantly inhibit complement activity even at thehighest concentration tested.

An experiment was performed to evaluate the effect of an anti-C5aantibody described herein (BNJ383) on classical pathway (CP) complementactivity in vitro using PNHS. The experiment utilized the Wieslab®Classical Pathway Complement Kit (Wieslab® COMPL CP310,Euro-Diagnostica, Sweden) and the associated protocol was followed withonly routine optimization well within the purview of theordinarily-skilled artisan. Briefly aliquots of the PNHS were incubatedin wells of a human IgM antibody-coated plate for one hour (at 37° C.)along with various concentrations (7.2, 3.6, 1.8, 0.9, 0.45, 0.2, 0.1,0.05, 0.02, or 0.01 μM) of an anti-hC5 antibody or an anti-hC5a antibody(BNJ383). The anti-C5 antibody inhibits the cleavage of human C5 intofragments C5a and C5b. As a control, several wells were incubated withPNHS under the same conditions, but in the absence of anti-hC5 antibodyor anti-hC5a antibody.

Following the incubation, the wells were washed thoroughly withkit-supplied 1× wash buffer. The level of alternative pathway complementactivation was measured by absorbance at 405 nm, following contact ofeach well with a kit-supplied enzyme conjugate (an anti-C5b-9 antibodyconjugated to alkaline phosphatase) and fluorogenic substrate (which isoperated upon by the enzyme) and incubation for 30 minutes at roomtemperature. The results are shown in FIG. 11.

While the anti-C5 antibody inhibited classical pathway complementactivity completely at concentrations greater than 0.1 μM, the anti-hC5aantibody did not significantly inhibit complement activity even at thehighest concentration tested.

Taken together, these results indicate that, in vitro, the anti-hC5aantibody, BNJ383, did not significantly affect C5b-9 generation(terminal complement activation) driven by either the classical oralternative pathway of complement, thus giving further evidence that theanti-hC5a antibodies described herein specifically target the free C5aanaphylatoxin arm of complement activation.

Example 17 Anti-C5a Antibody Retains an Unoccupied Antigen-Binding SiteAvailable to Bind to C5a Even in the Presence of a Molar Excess of hC5

The anti-C5a antibody BNJ383 (set forth above) was incubated at 4° C.for 84 hours in the presence of a 2.1-fold molar excess of human C5(hC5) to allow for complete antibody:C5 complex formation. A parallelexperiment was performed using an antibody that binds to human C5 at a2:1 stoichiometry (hereinafter the anti-C5 antibody). Antibody:C5complexes were resolved on a TSK™ G4000 SW size exclusion column (Tosoh,Tokyo) using a Waters™ 2690/5 HPLC system with a Waters™ W2487 dualwavelength detector to determine the occupancy of binding sites. Peakswere monitored at a wavelength of 214 nm. The mobile phase for the HPLCanalysis contained the following buffer composition: 3.9 mM NaH₂PO₄, 6.1mM Na₂HPO₄, and 150 mM NaCl, at pH7.0. The flow rate was 1.0 ml perminute and the run time was 20 minutes. Data were acquired and analyzedwith Waters Empower™ 2 chromatography software.

As depicted in FIG. 12, BNJ383 alone (FIG. 12A) and hC5 alone (FIG. 12B)each resolve as a single peak centered around 10.2 minutes and >95% ofthe anti-C5a antibody:hC5 complexes resolve as a single peak centeredaround 9.2 minutes (FIG. 12C). In contrast, complexes of hC5 and theanti-C5 antibody resolve in two peaks centered at 8.6 min (39%) and 9.2min (61%) (FIG. 12D). Thus, even in a molar excess of hC5, 95.2% of theBNJ383 has a free Fab arm that may be capable of binding to human C5a.

These samples were further examined to determine if the BNJ383 antibodyretained the ability to bind C5a in the presence of saturatingconcentrations of C5. Free antibody or antibody:C5 complexes weretitrated from 500 to 0.5 ng/mL on a streptavidin coated plate to whichbiotin conjugated hC5a was immobilized. Captured antibody was detectedwith an anti-human Fc antibody conjugated to horse radish peroxidase.The results depicted in FIG. 13 demonstrate that even BNJ383 complexedwith hC5 is capable of binding C5a and that the concentration ofantibody available to bind C5a is not detectably diminished in thepresence of saturating C5. Thus, the results described herein indicatethat the antibody, even in the presence of a molar excess of uncleavedC5, retains the ability to bind to free C5a with high affinity andthereby retains, even in that molar excess, the ability to inhibit thepro-inflammatory activity of C5a.

Example 18 BNJ383 is a Potent Antagonist of C5a but is anIncomplete/Partial Antagonist of Terminal Complement Complex FormationIn Vivo

Cynomolgus macaques were administered intravenously a single dose of theBNJ383 anti-C5a antibody at 1 mg/kg, 10 mg/kg, 100 mg/kg 250 mg/kg or400 mg/kg. Plasma samples were collected from the macaques at timepoints ranging from 1 day to 30 days following the administration of theantibody. Levels of C5a/C5a desarg in plasma were determined by anelectrochemiluminescent (ECL) assay in which a free C5a/C5a desarg wascaptured on a microtiter plate coated with an antibody specific for aneoepitope on C5a/C5a desarg and detected with a non-competitive C5aantibody conjugated to a ruthenate containing ECL moiety and read on aSECTOR 2400™ plate reader (MesoScale Discovery). Circulating antibodyconcentrations were determined by and enzyme linked immunosorbent assay(ELISA) in which free antibody (BNJ383) was captured on a microtiterplate coated with human C5a desarg and detected with a mouse anti-humanantibody conjugated to horseradish peroxidase (HRP).

As shown in FIG. 14, like the results described in Example 13,circulating concentrations of BNJ383 as low as 10 μg/mL deplete plasmaC5a/C5a desarg levels to below detectable limits in cynomolgus monkeys.These results also underscore that the antibody, even in the presence ofa molar excess of uncleaved C5, retains the ability to bind to free C5awith high affinity and thereby retains, even in that molar excess, theability to inhibit the pro-inflammatory activity of C5a.

To determine whether BNJ383 had an effect on hemolytic activity ofmacaque serum, the antibody was evaluated in an in vitro red blood cellhemolysis assay.

The red blood cell hemolysis assay is generally described in detail in,e.g., Rinder et al. (1995) J Clin Invest 96:1564-1572. Briefly, serumsamples obtained from macaques administered BNJ383 (as described above)were added to multiple wells of a 96 well assay plate such that theconcentration of the serum in each well was approximately 10%. The serumsamples, by virtue of the time points in which they were obtained,contained various concentrations of the BNJ383 antibody. The hemolyticactivity of serum from macaques not receiving BNJ383 served as anegative control and as the baseline hemolytic activity level.

Chicken erythrocytes (Lampire Biological Laboratories, Piperville, Pa.)were washed and resuspended in buffer at a final concentration of 5×10⁷cells/mL. The erythrocytes were sensitized to lysis by incubating thecells with an anti-chicken red blood cell polyclonal antibodycomposition. The sensitized erythrocytes were added to the wells of the96 well plate and the plate was incubated at 37° C. for 30 minutes.Hemoglobin release was measured by apparent absorbance at 415 nm using amicroplate reader.

As shown in FIG. 15A, even high concentrations of BNJ383 did notsubstantially inhibit erythrocyte hemolysis under these ex vivohemolytic assay experimental conditions.

The BNJ383 antibody was also evaluated to determine if it had an effecton complement activation of macaque serum using an ex vivo CH50eq assay.The CH50eq assay is a method for measuring the total classicalcomplement activity in serum. This test is an enzyme linkedimmunosorbent assay, which uses human gammaglobulins and mousemonoclonal antibodies as the activator of the classical complementpathway and captures the terminal complement complex (TCC) generated ona microtiter well coated with a TCC neoepitope specific antibody.Captured TCC is detected with a goat anti-TCC antibody conjugated tohorse radish peroxidase. The CH50eq assay provides a direct measure ofterminal complement complex (TCC) formation.

As shown in FIG. 15B, high concentrations of BNJ383 present in themacaque serum were capable of substantially inhibiting TCC formationunder these ex vivo conditions. These results indicate that the BNJ383antibody is not only capable of binding to and sequestering free C5a butis also capable of, as a function of concentration, partially orsubstantially inhibiting TCC formation.

An ex vivo experiment was also performed to evaluate the effect ofBNJ383 on classical pathway (CP) complement activity using the macaqueserum samples described above. The experiment utilized the Wieslab®Classical Pathway Complement Kit (Wieslab® COMPL CP310,Euro-Diagnostica, Sweden) and the associated protocol was followed withonly routine optimization well within the purview of theordinarily-skilled artisan. Briefly aliquots of the macaque serumsamples were incubated in wells of a human IgM antibody-coated plate forone hour. As a control, several wells were incubated under the sameconditions with serum from macaques not administered the BNJ383antibody.

Following the incubation, the wells were washed thoroughly withkit-supplied 1× wash buffer. The level of alternative pathway complementactivation was measured by absorbance at 405 nm, following contact ofeach well with a kit-supplied enzyme conjugate (an anti-C5b-9 antibodyconjugated to alkaline phosphatase) and fluorogenic substrate (which isoperated upon by the enzyme) and incubation for 30 minutes at roomtemperature. The results are shown in FIG. 15C.

The anti-hC5a antibody did significantly, though not completely, inhibitcomplement activity in a dose-dependent manner. Taken together, theresults described herein indicate that BNJ383 is not only a potentantagonist of C5a, but is also an incomplete/partial antagonist ofterminal complement complex formation in vivo. Thus, the antibody andantibodies sharing its properties are useful for treating a variety ofcomplement-associated disorders in which C5a-mediated inflammation isthe primary contributor to deleterious pathological effects and TCC mayplay a less significant or even beneficial role in the pathology.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

What is claimed is:
 1. An isolated nucleic acid comprising a nucleotidesequence encoding an antibody, or antigen binding fragment thereof,wherein the antibody, or antigen binding fragment thereof, comprises:(a) a light chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:140; a light chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:96; a light chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:142; a heavy chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:115; a heavy chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:144; and aheavy chain CDR3 comprising the amino acid sequence depicted in SEQ IDNO:117; (b) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:21; and a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:22; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:28; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:67;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:30; (c) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:156; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:157; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:158; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:160; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:161;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:162; (d) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:164; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:165; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:166; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:168; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:169;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:170; (e) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:172; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:173; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:174; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:176; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:177;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:178; (f) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:84; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:85; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:86; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:115; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:116;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:117; (g) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:88; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:89; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:90; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:119; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:120;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:121; (h) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:99; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:100; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:101; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:119; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:126;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:127; (i) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:21; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:113; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:28; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:46;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:47; (j) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:110; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:111; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:136; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:137;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:138; (k) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:105; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:106; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:107; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:115; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:124;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:117; (l) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:21; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:22; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:28; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:29;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:30; (m) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:38; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:22; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:28; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:29;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:30; (n) a light chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:20; a light chain CDR2 comprising the amino acidsequence depicted in SEQ ID NO:21; a light chain CDR3 comprising theamino acid sequence depicted in SEQ ID NO:22; a heavy chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:28; a heavychain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:46;and a heavy chain CDR3 comprising the amino acid sequence depicted inSEQ ID NO:47; or (o) a light chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:20; a light chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:38; a light chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:22; a heavychain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:28;a heavy chain CDR2 comprising the amino acid sequence depicted in SEQ IDNO:46; and a heavy chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:47.
 2. The isolated nucleic acid of claim 1,wherein the antibody, or antigen binding fragment thereof, comprises alight chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:20, a light chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:21, a light chain CDR3 comprising the amino acid sequencedepicted in SEQ ID NO:22, a heavy chain CDR1 comprising the amino acidsequence depicted in SEQ ID NO:28, a heavy chain CDR2 comprising theamino acid sequence depicted in SEQ ID NO:46, and a heavy chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:47.
 3. Anisolated nucleic acid comprising a nucleotide sequence encoding apolypeptide, wherein the polypeptide comprises the amino acid sequencedepicted in SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 36,SEQ ID NO: 40, SEQ ID NO: 44, or SEQ ID NO:
 49. 4. The isolated nucleicacid of claim 3, wherein the polypeptide comprises the amino acidsequence depicted in SEQ ID NO:
 40. 5. The isolated nucleic acid ofclaim 3, wherein the polypeptide comprises the amino acid sequencedepicted in SEQ ID NO:
 49. 6. An isolated nucleic acid comprising anucleotide sequence encoding an antibody variable region, wherein theantibody variable region is at least 90% identical to the amino acidsequence depicted in SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 37, SEQ IDNO: 42, or SEQ ID NO:
 45. 7. The isolated nucleic acid of claim 6,wherein the antibody variable region is at least 90% identical the aminoacid sequence depicted in SEQ ID NO:
 42. 8. The isolated nucleic acid ofclaim 6, wherein the antibody variable region is at least 90% identicalthe amino acid sequence depicted in SEQ ID NO:
 45. 9. An isolatednucleic acid comprising a nucleotide sequence encoding an antibodyvariable region, wherein the antibody variable region comprises theamino acid sequence depicted in SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO:37, SEQ ID NO: 42, or SEQ ID NO:
 45. 10. The isolated nucleic acid ofclaim 9, wherein the antibody variable region comprises the amino acidsequence depicted in SEQ ID NO:
 42. 11. The isolated nucleic acid ofclaim 9, wherein the antibody variable region comprises the amino acidsequence depicted in SEQ ID NO:
 45. 12. An isolated nucleic acidcomprising a nucleotide sequence encoding an antibody, or antigenbinding fragment thereof, wherein the antibody, or antigen bindingfragment thereof, comprises a light chain variable region comprising theamino acid sequence depicted in SEQ ID NO:42 and a heavy chain variableregion comprising the amino acid sequence depicted in SEQ ID NO:45. 13.An expression vector comprising the nucleic acid of claim
 1. 14. A hostcell comprising the nucleic acid of claim
 13. 15. An expression vectorcomprising the nucleic acid of claim
 3. 16. A host cell comprising thenucleic acid of claim
 15. 17. An expression vector comprising thenucleic acid of claim
 6. 18. A host cell comprising the nucleic acid ofclaim
 17. 19. An expression vector comprising the nucleic acid of claim9.
 20. A host cell comprising the nucleic acid of claim 19.